T-structure invasive cleavage assays, consistent nucleic acid dispensing, and low level target nucleic acid detection

ABSTRACT

The present invention relates to systems, methods and kits for low-level detection of nucleic acids, detecting at least two different viral sequences in a single reaction vessel, and increasing the dynamic range of detection of a viral target nucleic acid in a sample. The present invention also relates to T-structure invasive cleavage assays, as well as T-structure related target dependent non-target amplification methods and compositions. The present invention further relates to methods, compositions, devices and systems for consistent nucleic acid dispensing onto surfaces.

The present application is a continuation of pending U.S. patentapplication Ser. No. 11/811,544, filed Jun. 11, 2007, which will issueon Jul. 20, 2010 as U.S. Pat. No. 7,759,062, which claims priority toexpired U.S. Provisional Application Ser. No. 60/812,465, filed Jun. 9,2006, all of which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to systems, methods and kits for low-leveldetection of nucleic acids, detecting at least two different viralsequences in a single reaction vessel, and increasing the dynamic rangeof detection of a viral target nucleic acid in a sample. The presentinvention also relates to T-structure invasive cleavage assays, as wellas T-structure related target dependent non-target amplification methodsand compositions. The present invention further relates to methods,compositions, devices and systems for consistent nucleic acid dispensingonto surfaces.

BACKGROUND

All nucleic acid detection systems that rely on amplification of eitherthe target being detected or the signal being generated inherentlypossess a dynamic range that limits their usefulness. At lowconcentrations of the target being detected, the signal generated is toolow to detect or to low to be scored above background levels, andtherefore is below the limit of detection, i.e., outside the dynamicrange of the detection system. By contrast, at very high levels of thetarget being generated, the components of the detection system areexhausted such that the signal is said to be saturated, i.e. addition ofstill more target results in no increase in signal. In these cases, thequantity of target is said to be above the limit of detection, i.e.outside the dynamic range of the detection system.

In the real-world case of detection systems being used to detect targetsfrom biological specimens, the range of target present in the samplebeing detected can be quite large, and is often either below or abovethe limit of detection of the system in use. Therefore, previousattempts to cover larger ranges of target concentration have requiredthe generation of more than one detection system, to be used separately,that are optimized for a given dynamic range. Because the quantity oftarget nucleic acid in the specimen is by definition an unknownquantity, this very frequently requires the use of multiple detectionsystems sequentially to finally use the appropriate detection systemthat possesses the appropriate dynamic range for the specimen underexamination.

As such, a single detection system with a broader dynamic range, if itwas available, would significantly reduce costs, decrease labor time,and decrease expenditure of the specimen being examined. Even more, amethod of increasing the dynamic range of an existing detection systemwould greatly aid the field of detection of targets within biologicalspecimens generally.

SUMMARY OF THE INVENTION

The present invention provides systems, methods and kits for low-leveldetection of nucleic acids, detecting at least two different viralsequences in a single reaction vessel, and increasing the dynamic rangeof detection of a viral target nucleic acid in a sample. The presentinvention also provides T-structure invasive cleavage assays, as well asT-structure related target dependent non-target amplification methodsand compositions. The present invention further provides methods,compositions, devices and systems for consistent nucleic acid dispensingonto surfaces (e.g., through hydrophobic polymer dispensing componentsusing non-ionic detergents.

In some embodiments, the present invention provides methods fordetecting target nucleic acid in a sample, wherein the number of copiesof the target nucleic acid initially present in the sample is at lowcopy number (e.g., between about 2 and about 2000 copies or between 2and 100 copies), wherein the method comprises; a) incubating the samplewith a plurality of first and second probes, a plurality of reportersequences, and a cleavage agent under conditions such that: i) the firstprobes hybridize to first regions of the target nucleic and are cleavedby the cleavage agent thereby generating first 5′ cleaved portions, andii) the second probes hybridize to second regions of the target nucleicacid and are cleaved by the cleavage agent thereby generating second 5′cleaved portions; and b) detecting a signal from the reporter sequencesgenerated by hybridization of the first and second 5′ cleaved portionsto the reporter sequences thereby detecting the presence of the targetnucleic acid in the sample. In certain embodiments, the first and secondregions of the target nucleic acid are amplified prior to step a). Inother embodiments, the first and second regions of the target nucleicacid are amplified with at least 30 rounds of PCR prior to step a).

In particular embodiments, the present invention provides methods fordetecting target nucleic acid in a sample comprising; a) treating thesample with amplification reagents such that first and second regions ofthe target nucleic acids are amplified with at least 30 rounds of PCRthereby generating first and second amplified regions; b) incubating thesample with a plurality of first and second probes, a plurality ofreporter sequences, and a cleavage agent under conditions such that: i)the first probes hybridize to the first regions or the first amplifiedregions of the target nucleic and are cleaved by the cleavage agentthereby generating first 5′ cleaved portions, and ii) the second probeshybridize to the second regions or second amplified regions of thetarget nucleic acid and are cleaved by the cleavage agent therebygenerating second 5′ cleaved portions; and c) detecting any signal fromthe reporter sequences generated by hybridization of the first andsecond 5′ cleaved portions to the reporter sequences thereby detectingthe presence or absence of the target nucleic acid in the sample.

In some embodiments, the number of copies of the target nucleic acidinitially present in the sample is between about 2 and about 100 copies,and the presence of the target nucleic acid is detected in the sample.In further embodiments, the number of copies of the target nucleic acidinitially present in the sample is between about 2 and about 10 copies,and the presence of the target nucleic acid is detected in the sample.In certain embodiments, the amplification reagents comprise a firstprimer pair for the first region of the target nucleic acid and a secondprimer pair for the second region of the target nucleic acid. In otherembodiments, the first primer pair comprises a first forward primer anda first reverse primer, and wherein either the first forward primer orthe first reverse primer is configured to also serve as an upstreamprobe such that it can form an invasive cleavage structure with thefirst probe and the first amplified region. In further embodiments, thesecond primer pair comprises a second forward primer and a secondreverse primer, and wherein either the second forward primer or thesecond reverse primer is configured to also serve as an upstream probesuch that it can form an invasive cleavage structure with the secondprobe and the second amplified region.

In particular embodiments, the target nucleic acid is viral nucleicacid. In other embodiments, the viral nucleic acid comprises at least aportion of a RNA viral genome. In further embodiments, the viral nucleicacid is from hepatitis C virus. In some embodiments, the signal isgenerated by cleavage of the reporter sequences. In other embodiments,the first and second probes are cleaved as part of an invasive cleavagereaction.

In some embodiments, the incubating further comprises incubating thesample with first upstream oligonucleotides configured to form invasivecleavage structures with the first probes and the first amplifiedregions. In certain embodiments, the incubating further comprisesincubating the sample with second upstream oligonucleotides configuredto form invasive cleavage structures with the second probes and thesecond amplified regions.

In particular embodiments, the first and second regions of the targetnucleic acid are non-overlapping. In other embodiments, the reportersequences comprise a dye and a quencher. In some embodiments, the firstand second 5′ cleaved portions are identical to each other.

In additional embodiments, the methods further comprise incubating thesample with a plurality control target sequences and a plurality ofthird probes designed to detect the presence of the control sequences.In other embodiments, the first and second 5′ cleaved portions areconfigured to not hybridize to the first and second regions, or thefirst and second amplified regions, of the target nucleic acid.

In some embodiments, the present invention provides compositions, kits,and methods of quantitating viral nucleic acid targets using multipleprobes that bind to a viral target nucleic acid at different strengths.In some embodiments, groups of probes are used in which each probeexhibits different binding affinities to the viral target sequence(e.g., by altering complementarity, length, concentration, additives,etc.). The use of multiple probes with different properties allows foran increase in the dynamic range of detection assays. In someembodiments, the multiple probes are used in invasive cleavage assays.

Accordingly, in some embodiments, the present invention provides amethod for detecting the presence of, absence of, or amount of a viraltarget nucleic acid in a sample, comprising: incubating a samplesuspected of containing a viral target nucleic acid with a plurality offirst probe oligonucleotides and a plurality of second probeoligonucleotides, wherein each of the first and second probeoligonucleotides comprises an analyte specific region, wherein theplurality of second probe oligonucleotides are configured to occupy aprobe hybridization site on the viral target nucleic acid at a differentfrequency than the plurality of first probe oligonucleotides; andmeasuring hybridization of the first and said second probeoligonucleotides over time, thereby measuring the amount of the viraltarget nucleic acid. In some embodiments a plurality of third, fourth,fifth, etc. probe oligonucleotides are used. These additionaloligonucleotides may be configured to bind to the same analyte-specificregion of a viral target nucleic acid or may bind to differentanalyte-specific regions of the same or different target nucleic acids(e.g., the third and fourth probes are configured to hybridize to asecond analyte-specific region of the same viral target nucleic acidsuch that the third probe occupies the hybridization site at a differentfrequency than the fourth probe).

In some embodiments, the analyte specific regions of the first probeoligonucleotides are completely complementary to the viral targetnucleic acid. In some embodiments, the analyte specific regions of thesecond probe oligonucleotides are partially complementary to the viraltarget nucleic acid (e.g., contain a single mismatch). In someembodiments, the second probe oligonucleotide is shorter in length thanthe first probe oligonucleotide (e.g., by one, two, three, or four ormore nucleotides). In some embodiments, the second probeoligonucleotides are present at least a 5 fold, and preferably at leasta 10 fold lower concentration than the first probe oligonucleotides. Insome embodiments, the second probe oligonucleotides are present at leasta 20 fold (e.g., 100 fold, 500 fold, 1000 fold, 10,000 fold, etc. lowerconcentration than the first probe oligonucleotide). Where three or moreprobes of different concentrations are used, each probe may be separatedby at least 5 fold (10 fold, 20 fold, 100 fold, etc.) concentration fromone another (e.g., a third probe 10000 fold more than a first probe anda second probe 100 fold more than a first probe). In some embodiments,one of the mixtures comprises an agent known to increase or decreasehybridization efficiency (e.g., a charge tag, minor groove bindingagent, or an intercalating agent). In other embodiments, one of theprobes comprises one or more modified bases (e.g., amino T, indole, ornitropyrrole). In some embodiments, the analyte specific region ofsecond probe oligonucleotide is shorter than the analyte specific regionof the first probe oligonucleotide (e.g., by one or more nucleotides).In other embodiments, the analyte specific region of the second probeoligonucleotide comprises increased secondary structure relative to theanalyte specific region of the first probe oligonucleotide. In certainembodiments, the first probe oligonucleotides further comprise anon-analyte specific region, wherein the non-analyte specific regioncomprises one or more nucleotides that are not complementary to theviral target nucleic acid. In some embodiments, each of the second probeoligonucleotides further comprises a non-analyte specific region,wherein the non-analyte specific region comprises one or morenucleotides that are not complementary to the viral target nucleic acid.In some embodiments, incubating the sample with the second probeoligonucleotides comprises incubating the sample with competitoroligonucleotides, wherein the competitor oligonucleotides each comprisea region that is complementary to the non-analyte specific regions ofthe second probe oligonucleotides. The present invention is not limitedby the nature of the competitor. The competitor may be a second viraltarget nucleic acid or a different region of the first oligonucleotidewhere, for example, hybridization of the non-analyte specific region ofthe second probe to the competitor does not generate a detectable eventor generates a detectable event that is distinguishable from thedetectable event generated by the first and/or second probes hybridizingto the analyte-specific region.

In some embodiments, incubating the sample with the second probeoligonucleotides comprises incubating the sample with competitoroligonucleotides, wherein the competitor oligonucleotides each comprisea region that is complementary to the non-analyte specific regions ofthe second probe oligonucleotides. In certain embodiments, one of themixtures comprises altered reaction conditions that alter hybridizationefficiency of a probe (e.g., altered pH, buffer, ionic strength oradditional compositions (e.g., crowding agents)).

In some embodiments, the sample is a sample from an animal (e.g., ahuman) comprising blood, serum, stool, urine, or lymph known to orsuspected of comprising a target nucleic acid (e.g., a virus or abacterium). In some embodiments, the sample comprises a purified sampleof nucleic acid (e.g., total DNA or RNA from a tissue, fluid or cell;genomic DNA; etc.). In some embodiments, the viral target nucleic acidis from human immunodeficiency virus (HIV) and other retroviruses,hepatitis C virus (HCV), hepatitis B virus (HBV), hepatitis A virus(HAV), human cytomegalovirus, (CMV), herpes simplex virus (HSV), Epsteinbar virus (EBV), varicella zoster virus (VZV), human papilloma virus(HPV), bacteriophages (e.g., phage lambda), influenzaviruses,adenoviruses, or lentiviruses) or a bacterium (e.g., Chlamydia sp., N.gonorrhea, or group B streptococcus). In other embodiments, the sampleis from a plant. For example, in some embodiments, the plant is infectedwith or suspected of being infected with a virus.

In some embodiments, the methods of incubating the sample with the firstand second probe oligonucleotides occur in the same reaction vessel(e.g., the first and second probe oligonucleotides are mixed in solutionin the same reaction vessel). In some embodiments, the first an secondprobe oligonucleotides comprise labels. In some embodiments, the firstand second labels are different from each other. In some embodiments,the first and second labels are the same label. In some embodiments,measuring the hybridization of the first and second probeoligonucleotides comprises performing an invasive cleavage structuretype assay (e.g., an INVADER assay). In some such embodiments, theprobes are unlabeled, but comprise a flap sequence that is removed fromthe probe upon cleavage during the invasive cleavage assay. In someembodiments, the removed flaps are configured to hybridize to a FRETcassette to trigger a detection reaction. In some embodiments, the firstand second probes report to the same FRET cassette (e.g., the first andsecond probe generate identical flaps upon cleavage in the primaryinvasive cleavage reaction). In other embodiments, determining theamount of the target nucleic acid comprises performing a detection assayincluding, but not limited to, a hybridization assay, any real-timeamplification assay that involves hybridization, a TAQMAN assay, SNP-ITassay, a Southern blot, a ligase assay, a microarray assay, aFULLVELOCITY assay, a cycling probe assay, NASBA, branched DNA assay,TMA, methods employing molecular beacons, capillary electrophoresisdetection methods, microfluidic detection methods, and the like.

In other embodiments, the present invention provides a method fordetecting the presence of, absence of, or amount of a viral targetnucleic acid in a sample, comprising: providing a sample containing orsuspected of containing a viral target nucleic acid; a first probeoligonucleotide comprising an analyte specific region and a first label,wherein the analyte specific region of the first probe oligonucleotideis completely complementary to the viral target nucleic acid; and asecond probe oligonucleotide comprising an analyte specific region and asecond label, wherein the analyte specific region of the second probeoligonucleotide is partially complementary to the viral target nucleicacid; and exposing the sample to the first and second probeoligonucleotides; and, in some embodiments, determining the amount ofthe viral target nucleic acid in the sample.

The present invention further provides a kit comprising reagents and, insome embodiments, instructions, for performing the detection assays ofthe present invention. For example, in some embodiments, the presentinvention provide a kit for detecting the presence of, absence of, orquantitation of viral target nucleic acids in a sample, comprising: aplurality of first probe oligonucleotides comprising a first analytespecific region and, optionally, a first label, and a plurality ofsecond probe oligonucleotides comprising a second analyte specificregion and, optionally, a second label, wherein the second probeoligonucleotides are configured to occupy a probe hybridization site onthe viral target nucleic acid at a different frequency than the firstmixture of probe oligonucleotides; and reagents for performing anINVADER assay using the first and second probe oligonucleotides. In someembodiments, the analyte specific regions of the first probeoligonucleotides are completely complementary to the viral targetnucleic acid. In other embodiments, the analyte specific regions of thesecond probe oligonucleotides are partially complementary to the viraltarget nucleic acid (e.g., contain one or more mismatches with the viraltarget nucleic acid). In still further embodiments, the second probeoligonucleotides are present at a lower concentration than the firstprobe oligonucleotides. In some embodiments, the kit further comprisesinstructions for using the kit for performing a nucleic acid detectionassay. In some embodiments, the kit comprises reagents and/orinstructions for use of the methods of the present invention with a oneor more different detection assay technologies (e.g., an invasivecleavage assay (e.g., INVADER assay), a TAQMAN assay, SNP-IT assay,etc.)).

In some embodiments, the present invention provides methods fordetecting a viral target nucleic acid, comprising: a) amplifying a viraltarget nucleic acid at two different levels of amplification to generateamplification products; b) hybridizing the amplification products to afirst probe and second probe, wherein the first probe hybridizes to theamplification products at a different frequency than the second probe.In certain embodiments, the second probe is present at a 10-fold lowerconcentration than the first probe. In other embodiments, the at leasttwo probes bind to the same sequence.

In additional embodiments, the present invention provides methods fordetecting a viral target nucleic acid in a plurality of samples over abroad dynamic range, comprising: exposing a first sample having lessthan 10̂3 copies of viral target nucleic acid and a second sample havinggreater than 10̂5 copies of viral target nucleic acid to a set ofreagents under conditions such that the viral target nucleic acid in thefirst and second samples is detected, wherein method comprises exposingeach of the first and second samples to a first probe and a secondprobe, wherein the second probe hybridizes to the viral target nucleicacids at a different frequency than the first probe. In particularembodiments, the viral target nucleic acid in the first and secondsamples is quantitated. In further embodiments, the second probe ispresent at a 10-fold lower concentration than the first probe. In someembodiments, the viral target nucleic acids are treated under two ormore different amplification conditions prior to detection. In otherembodiments, the method is conducted without any amplification of theviral target nucleic acid.

In some embodiments, the present invention provides methods fordetecting a viral target nucleic acid, comprising: a) amplifying a viraltarget nucleic acid to generate amplification products; b) contactingthe amplification products with first and second probes, wherein thesecond probe hybridizes to the amplification products at a differentfrequency that the first probe; c) cleaving the first and second probes;and d) detecting the cleavage of the first and second probes.

In other embodiments, the present invention provides kits comprising: apolymerase, a 5′ nuclease, and two probes configured to hybridize to ananalyte-specific region of a viral target nucleic acid, wherein thesecond probe hybridizes to the analyte-specific region at a differentfrequency than the first probe oligonucleotide, and wherein the firstand second probes are configured to both directly or indirectly generatea detectable signal in the presence of the viral target nucleic acid. Insome embodiments, the first and second probes generate the same type ofdetectable signal. In certain embodiments, the first and second probeseach comprise a flap sequence that is complementary to a FRET cassette.In other embodiments, the flap of the first probe is identical to theflap of the second probe.

In some embodiments, the present invention provides methods fordetecting a viral target nucleic acid in a sample comprising; a)contacting a sample suspected of containing a viral target nucleic acidwith amplification reagents such that, if the viral target nucleic acidis present: i) a first region of the viral target nucleic acid is eithernot amplified, or is amplified at a first level to generate plurality offirst product sequences; and ii) a second region of the viral targetnucleic acid is amplified at a second level to generate a plurality ofsecond product sequences, wherein the second level of amplification isgreater than the first level of amplification (e.g. such that the secondproduct sequences are present at a level of at least 10-fold . . .100-fold . . . 1000-fold . . . 10,000-fold . . . or 100,000-fold higherconcentration after amplification that the target nucleic acid, or firstproduct sequences if produced); and b) incubating the sample with aplurality of first and second probe oligonucleotides, wherein: i) thefirst and second probe oligonucleotides hybridize to the first region ofthe target nucleic acid, and the first product sequences is produced, atdifferent frequencies, or ii) the first and second probeoligonucleotides hybridize to the second product sequences at adifferent frequency; and c) measuring hybridization of the first andsecond probe oligonucleotides thereby detecting the target nucleic acidin the sample. In particular embodiments, the second product sequencesare present at a level between 100-fold and 100,000 fold higherconcentration after amplification than the target nucleic acid, or firstproduct sequences if produced.

In certain embodiments, the present invention provides methods fordetecting a viral target nucleic acid in a plurality of samples over abroad dynamic range, comprising: exposing a first sample having lessthan 10³ copies of viral target nucleic acid and a second sample havinggreater than 10⁵ copies of viral target nucleic acid to a set ofreagents under conditions such that the viral target nucleic acid in thefirst and second samples is detected, wherein the method comprisesexposing each of the first and second samples to a first probe and asecond probe, wherein the second probe hybridize to the viral target atdifferent frequencies.

In particular embodiments, the present invention provides methods fordetecting a viral target nucleic acid, comprising: a) linearlyamplifying a first region of the viral target nucleic acid to generatelinearly amplified amplification products; b) exponentially amplifying asecond region of the viral target nucleic acid to generate exponentiallyamplified amplification products; c) hybridizing the linearly amplifiedamplification products with a first set of probes and the exponentiallyamplified amplification products with a second set of probes, whereineither the first or the second set of probes comprises a first pluralityof probes that hybridize to amplified viral target nucleic acid and asecond plurality of probes that hybridize to amplified viral targetnucleic acid at a different frequency than the first plurality ofprobes. In certain embodiments, both the first set and the second set ofprobes comprises a first plurality of probes that hybridize to amplifiedviral target nucleic acid and a second plurality of probes thathybridize to amplified viral target nucleic acid at a differentfrequency than the first plurality of probes.

In some embodiments, the present invention provides methods fordetecting a viral target nucleic acid, comprising: a) amplifying a viraltarget nucleic acid both linearly and exponentially to generateamplification products; b) hybridizing the amplification products to atleast two probes, wherein the first probe hybridizes to amplified viraltarget nucleic acid at a different frequency than the second probe. Incertain embodiments, the first and second probes both hybridize to thesame probe binding site on the viral target nucleic acid.

In certain embodiments, the present invention provides methods fordetecting a viral target nucleic acid in a sample comprising; a)contacting a sample suspected of containing a viral target nucleic acidwith amplification reagents such that, if the viral target nucleic acidis present: i) a first region of the viral target nucleic acidcomprising a first probe hybridization site is either not amplified, oris amplified at a first level to generate plurality of first productsequences that comprise the first probe hybridization site; and ii) asecond region of the viral target nucleic acid is amplified at a secondlevel to generate a plurality of second product sequences that comprisea second probe hybridization site, wherein the second level ofamplification is greater than the first level of amplification (e.g.such that the second product sequences are present at a level of atleast 10-fold . . . 100-fold . . . 1000-fold . . . 10,000-fold . . . or100,000-fold higher concentration after amplification that the viraltarget nucleic acid, or first product sequences if produced); and b)incubating the sample with a plurality of first and second probeoligonucleotides, wherein: i) the first and second probeoligonucleotides occupy the first probe hybridization site on the firstregion of the viral target nucleic acid, and the first product sequencesif produced, at different frequencies, or ii) the first and second probeoligonucleotides occupy the second probe hybridization site on thesecond product sequences at a different frequency; and c) measuringhybridization of the first and second probe oligonucleotides therebydetecting the viral target nucleic acid in the sample. In particularembodiments, the second product sequences are present at a level between100-fold and 100,000 fold higher concentration after amplification thanthe viral target nucleic acid, or first product sequences if produced.

In other embodiments, the methods further comprise incubating the samplewith a third probe oligonucleotide that occupies the first probehybridization site on the first region of the viral target nucleic acid,and the first product sequences if produced, at a first frequency, andmeasuring the hybridization of the third probe oligonucleotide. In someembodiments, the methods further comprise incubating the sample with afourth probe oligonucleotide that occupies the first probe hybridizationsite on the first region of the viral target nucleic acid, and the firstproduct sequences if produced, at a second frequency, wherein the secondfrequency is different from the first frequency, and measuring thehybridization of the fourth probe oligonucleotide.

In certain embodiments, the methods further comprise incubating thesample with a third probe oligonucleotide that occupies the second probehybridization site on the second product sequences at a first frequency,and measuring the hybridization of the third probe oligonucleotide. Inparticular embodiments, the methods further comprise incubating thesample with a fourth probe oligonucleotide that occupies the secondprobe hybridization site on the second product sequences at a secondfrequency, wherein the second frequency is different from the firstfrequency, and measuring the hybridization of the fourth probeoligonucleotide.

In some embodiments, the first level of amplification is achieved bylinear amplification, and the second level is achieved is achieved withlogarithmic amplification (e.g., polymerase chain reaction). In furtherembodiments, the first level of amplification is achieved withcompromised amplification (e.g. using inefficient primers and/orinefficient polymerases). In other embodiments, the second level ofamplification is at least 10-fold greater than no amplification or thefirst level of amplification.

In some embodiments, the present invention provides methods fordetecting a viral target nucleic acid in a sample, comprising; a)contacting a sample suspected of containing a viral target nucleic acidwith amplification reagents such that, if the viral target nucleic acidis present: i) a first region of the viral target nucleic acid isamplified non-logarithmically to generate a plurality ofnon-logarithmically amplified sequences that comprise a first probehybridization site, and ii) a second region of the viral target nucleicacid is amplified logarithmically to generate a plurality oflogarithmically amplified sequences that comprise a second probehybridization site; b) incubating the sample with a plurality of firstprobe oligonucleotides, a plurality of second probe oligonucleotides,and a plurality of third probe oligonucleotides, wherein each of thefirst, second, and third probe oligonucleotides comprises an analytespecific region, wherein the plurality of second probe oligonucleotidesare configured to occupy the second probe hybridization site on thelogarithmically amplified sequences at a different frequency than theplurality of first probe oligonucleotides, and wherein the third probeoligonucleotides are configured to occupy the first probe hybridizationsite on the non-logarithmically amplified sequences at a firstfrequency; and c) measuring hybridization of the first, second, andthird probe oligonucleotides, thereby detecting the viral target nucleicacid in the sample. In other embodiments, the viral target nucleic acidis initially present in the sample in an amount between about 10¹ andabout 10⁸ molecules (e.g. the dynamic range of the methods extend overat least about seven orders of magnitude).

In certain embodiments, the measuring detects the amount of the viraltarget nucleic acid in the sample. In other embodiments, the measuringis conduced over time. In further embodiments, the plurality oflogarithmically amplified sequences do not contain the first probehybridization site.

In particular embodiments, the analyte specific regions of the firstprobe oligonucleotides are completely complementary to the second probehybridization site of the second product sequence (e.g. logarithmicallyamplified sequences). In other embodiments, the analyte specific regionsof the second probe oligonucleotides are partially complementary to thesecond probe hybridization site of the second product sequences (e.g.,logarithmically amplified sequences).

In certain embodiments, the methods further comprise incubating thesample with a plurality of fourth probe oligonucleotides comprising ananalyte specific region, wherein the fourth probe oligonucleotides areconfigured to occupy the first probe hybridization site on the firstproduct sequences (e.g., non-logarithmically amplified sequences) at asecond frequency which is different from the first frequency of thethird probe oligonucleotides. In further embodiments, the viral targetnucleic acid is initially present in the sample in an amount betweenabout 10¹ and about 10¹⁰ molecules (e.g. the dynamic range of themethods extend over at least about nine orders of magnitude).

In further embodiments, the analyte specific regions of the third probeoligonucleotides are completely complementary to the first probehybridization site of the first product sequences (e.g.,non-logarithmically amplified sequences). In other embodiments, theanalyte specific regions of the third probe oligonucleotides arepartially complementary to the first probe hybridization site of thefirst product sequences (e.g, non-logarithmically amplified sequences).In additional embodiments, the analyte specific regions of the thirdoligonucleotides are identical to the analyte specific regions of thefourth oligonucleotides.

In some embodiments, the second probe oligonucleotides are present in atleast a 5-fold lower concentration than the first probe oligonucleotides(e.g. 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold lower concentration). Incertain embodiments, the second probe oligonucleotides are present in atleast a 10-fold lower concentration than the first probeoligonucleotides (e.g. 10-fold . . . 15-fold . . . 25-fold . . . 50-fold. . . 75-fold . . . or 95-fold lower concentration, or any range between10-fold and 100-fold). In particular embodiments, the second probeoligonucleotides are present in at least a 100-fold lower concentrationthan the first probe oligonucleotides (e.g. 100-fold . . . 125-fold . .. 150-fold . . . 250-fold . . . 500-fold . . . 750-fold . . . or900-fold lower concentration, or any range between 100-fold and1000-fold). In further embodiments, the second probe oligonucleotidesare present in at least a 1000-fold lower concentration than the firstprobe oligonucleotides (e.g., 1000-fold . . . 1100-fold . . . 1300-fold. . . 1500-fold . . . 10,000-fold . . . 15,000-fold . . . 25,000-fold .. . 100,000-fold . . . 500,000-fold . . . or 1,000,000-fold, or anyrange between 1000-fold and 1,000,000-fold).

In some embodiments, the third probe oligonucleotides are present in atleast a 5-fold lower concentration than the fourth probeoligonucleotides (e.g. 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold lowerconcentration). In certain embodiments, the third probe oligonucleotidesare present in at least a 10-fold lower concentration than the fourthprobe oligonucleotides (e.g. 10-fold . . . 15-fold . . . 25-fold . . .50-fold . . . 75-fold . . . or 95-fold lower concentration, or any rangebetween 10-fold and 100-fold). In particular embodiments, the thirdprobe oligonucleotides are present in at least a 100-fold lowerconcentration than the fourth probe oligonucleotides (e.g. 100-fold . .. 125-fold . . . 150-fold . . . 250-fold . . . 500-fold . . . 750-fold .. . or 900-fold lower concentration, or any range between 100-fold and1000-fold). In further embodiments, the third probe oligonucleotides arepresent in at least a 1000-fold lower concentration than the fourthprobe oligonucleotides (e.g., 1000-fold . . . 1100-fold . . . 1300-fold. . . 1500-fold . . . 10,000-fold . . . 15,000-fold . . . 25,000-fold .. . 100,000-fold . . . 500,000-fold . . . or 1,000,000-fold, or anyrange between 1000-fold and 1,000,000-fold).

In certain embodiments, the viral target nucleic acid is initiallypresent in the sample in an amount between about 10¹ and about 10³molecules, and the amount of the viral target nucleic acid is determinedby the measuring hybridization of the first probe oligonucleotides. Inother embodiments, the viral target nucleic acid is initially present inthe sample in an amount between about 10³ and about 10⁶ molecules, andthe amount of the viral target nucleic acid is determined by themeasuring hybridization of the second probe oligonucleotides. In someembodiments, the viral target nucleic acid is initially present in thesample in an amount between about 10⁶ and about 10⁸ molecules, and theamount of the viral target nucleic acid is determined by the measuringhybridization of the third probe oligonucleotides.

In certain embodiments, the method is conducted on two samples, whereinthe viral target nucleic acid is initially present in one sample in anamount less than 10³ and initially present in a second sample in anamount greater than 10⁵. In other embodiments, the method is conductedon two samples, wherein the viral target nucleic acid is initiallypresent in one sample in an amount less than 10² and initially presentin a second sample in an amount greater than 10⁶. In furtherembodiments, the method is conducted on two samples, wherein the viraltarget nucleic acid is initially present in one sample in an amount lessthan 10¹ and initially present in a second sample in an amount greaterthan 10⁷, or greater than 10⁸, or greater than 10⁹.

In particular embodiments, the plurality of first product sequences(e.g, non-logarithmically amplified sequences) further comprise thesecond probe hybridization site. In other embodiments, the plurality ofsecond product sequences (e.g., non-logarithmically amplified sequences)do not contain the second probe hybridization site. In certainembodiments, the non-logarithmic amplification of the first regioncomprises single-stranded PCR or compromised PCR.

In some embodiments, the first probe oligonucleotides further comprise anon-analyte specific region, wherein the non-analyte specific regioncomprises one or more nucleotides that are not complementary to thesecond product sequences (e.g, logarithmically amplified sequences). Inother embodiments, the second probe oligonucleotides further comprise anon-analyte specific region, wherein the non-analyte specific regioncomprises one or more nucleotides that are not complementary to thesecond product sequences (e.g., logarithmically amplified sequences). Inother embodiments, the third probe oligonucleotides further comprise anon-analyte specific region, wherein the non-analyte specific regioncomprises one or more nucleotides that are not complementary to thefirst product sequences (e.g, non-logarithmically amplified sequences).In further embodiments, the fourth probe oligonucleotides furthercomprise a non-analyte specific region, wherein the non-analyte specificregion comprises one or more nucleotides that are not complementary tothe first product sequences (e.g, non-logarithmically amplifiedsequences).

In certain embodiments, the analyte specific region of second probeoligonucleotide is shorter than the analyte specific region of the firstprobe oligonucleotide. In other embodiments, the analyte specific regionof the fourth probe oligonucleotide is shorter than the analyte specificregion of the third probe oligonucleotide.

In some embodiments, the first probe oligonucleotides comprise firstlabels and wherein the second probe oligonucleotides comprise secondlabels. In other embodiments, the third probe oligonucleotides comprisethird labels and the fourth probe oligonucleotides comprise fourthlabels. In particular embodiments, at least one of the first, second, orthird oligonucleotides is unlabeled. In additional embodiments, thefirst, second, and third probe oligonucleotides are unlabeled. In someembodiments, the fourth probe oligonucleotides are un-labeled. Incertain embodiments, the fourth probe oligonucleotides comprises alabel. In other embodiments, the first, the second, and the third labelsare different from each other or are the same as each other. In certainembodiments, the amplification reagents comprise first and secondprimers, and a polymerase.

In some embodiments, the first and second probe oligonucleotides furthercomprise a non-analyte specific region configured to not hybridize tothe second probe hybridization site of the second product sequences(e.g, logarithmically amplified sequences), wherein the non-analytespecific region is 5′ of the analyte specific region. In certainembodiments, the first and second probe oligonucleotides form aninvasive cleavage structure with an upstream oligonucleotide, whereinthe upstream oligonucleotide comprise a 5′ portion and a 3′ portion,wherein the 5′ portion is configured to hybridize to a region contiguouswith the second probe hybridization site on the second product sequences(e.g., logarithmically amplified sequences), and wherein the 3′ portionis configured to not hybridize to the second product sequences (e.g.,logarithmically amplified sequences). In other embodiments, the methodsfurther comprise incubating the sample with a plurality of additionalprobe oligonucleotides comprising an analyte specific region, whereinthe additional probe oligonucleotide is configured to occupy the secondprobe hybridization site on the second product sequences (e.g.,logarithmically amplified sequences) at a frequency different that thefirst and second probe oligonucleotides.

In particular embodiments, the present invention provides methods fordetecting an amount of a viral target nucleic acid in a sample,comprising; a) incubating a sample suspected of containing a viraltarget nucleic acid with a plurality of first probe oligonucleotides anda plurality of second probe oligonucleotides, wherein each of the firstand the second probe oligonucleotides comprises an analyte specificregion, wherein the plurality of second probe oligonucleotides areconfigured to occupy a probe hybridization site on the viral targetnucleic acid with the same affinity as the plurality of first probeoligonucleotides, and wherein the plurality of second probeoligonucleotides are present in at least 5-fold lower concentration thanthe first probe oligonucleotides; and b) measuring hybridization of thefirst and the second probe oligonucleotides over time, thereby detectingthe amount of the viral target nucleic acid. In some embodiments, thefirst probe oligonucleotides further comprise a first non-analytespecific region, and the second probe oligonucleotides further comprisea second non-analyte specific region which is not identical to the firstnon-analyte specific region. In other embodiments, the analyte specificregions of the first and second oligonucleotides have an identicalsequence.

In additional embodiments, the present invention provides methods fordetecting an amount of a viral target nucleic acid in a sample,comprising; a) incubating a sample suspected of containing a viraltarget nucleic acid with a plurality of un-labeled first probeoligonucleotides and a plurality of un-labeled second probeoligonucleotides, wherein each of the first and the second probeoligonucleotides comprises an analyte specific region, wherein theplurality of second probe oligonucleotides are configured to occupy aprobe hybridization site on the viral target nucleic acid at a differentfrequency than the plurality of first probe oligonucleotides; and b)measuring hybridization of the first and the second probeoligonucleotides over time, thereby detecting the amount of the viraltarget nucleic acid.

In further embodiments, the present invention provides methods fordetecting an initial amount of a viral target nucleic acid in a samplewithout amplifying initial amount of the viral target nucleic acid,comprising; a) incubating a sample initially containing 300 copies orless of a viral target nucleic acid with a plurality of first probeoligonucleotides and a plurality of second probe oligonucleotides,wherein each of the first and the second probe oligonucleotidescomprises an analyte specific region, wherein the plurality of secondprobe oligonucleotides are configured to occupy a probe hybridizationsite on the viral target nucleic acid at a different frequency than theplurality of first probe oligonucleotides; b) measuring hybridization ofthe first and the second probe oligonucleotides over time, therebymeasuring the amount of the viral target nucleic acid, wherein the 300copies or less of the viral target nucleic acid are not amplified priorto the measuring step. In particular embodiments, the 300 copies or lessis between 100 and 300 copies or between 100 and 200 copies.

In some embodiments, the present invention provides methods fordetecting an amount of a viral target nucleic acid in a sample,comprising; a) contacting a sample suspected of containing viral targetnucleic acid with amplification reagents such that, if the viral targetnucleic acid is present, a region of the viral target nucleic acidcontaining a probe hybridization site is amplified to generate aplurality of amplified sequences, b) incubating the sample with aplurality of first probe oligonucleotides and a plurality of secondprobe oligonucleotides, wherein each of the first and the second probeoligonucleotides comprises an analyte specific region, wherein theplurality of second probe oligonucleotides are configured to occupy thea probe hybridization site on the amplified sequence at a differentfrequency than the plurality of first probe oligonucleotides; c)measuring hybridization of the first and the second probeoligonucleotides over time, thereby measuring the amount of the viraltarget nucleic acid, wherein the measuring is possible when the viraltarget nucleic acid is initially present in the sample in an amountbetween about 1 molecule and about 10⁷ molecules.

In certain embodiments, the incubating and measuring steps are conductedin a single vessel. In other embodiments, the contacting, incubating,and measuring steps are conducted in a single vessel. In furtherembodiments, the analyte specific regions of the first oligonucleotidesare identical to the analyte specific regions of the secondoligonucleotides. In some embodiments, the analyte specific regions ofthe second probe oligonucleotides contain a single mismatch with thelogarithmically amplified sequences.

In some embodiments, the second or fourth probe oligonucleotides containa charge tag. In other embodiments, the second or fourth probeoligonucleotide contains at least one modified nucleotide. In furtherembodiments, the second probe oligonucleotide has a lower or higheraffinity for the second probe hybridization site than the first probeoligonucleotide. In particular embodiments, the second probeoligonucleotide has a lower or higher Tm with the second probehybridization site than the first probe oligonucleotide. In additionalembodiments, the fourth probe oligonucleotide has a lower or higheraffinity for the first probe hybridization site than the third probeoligonucleotide. In other embodiments, the fourth probe oligonucleotidehas a lower or higher Tm with the first probe hybridization site thanthe third probe oligonucleotide.

In some embodiments, the measuring hybridization of the first, second,and/or third, and/or fourth probe oligonucleotides comprises performinga hybridization assay. In particular embodiments, the hybridizationassay is selected from the group consisting of a TAQMAN assay, SNP-ITassay, an invasive cleavage assay, a Southern blot, and a microarrayassay. In further embodiments, the invasive cleavage assay in an INVADERassay.

In certain embodiments, the present invention provides methods forgenotyping a polymorphic locus in a viral target nucleic acid in asample, comprising; a) contacting a sample suspected of containing theviral target nucleic acid with amplification reagents such that, if theviral target nucleic acid is present, a region of the viral targetnucleic acid containing the polymorphic locus is amplified to generate aplurality of amplified sequences, wherein the amplification is conducteduntil saturation; b) incubating the sample with a plurality of firstprobe oligonucleotides and a plurality of second probe oligonucleotides,wherein each of the first probe oligonucleotides comprises: i) a firstanalyte specific region configured for detecting a first allele at thepolymorphic locus, and ii) a label capable of generating a detectablesignal or a cleavable portion configured to cause a detectable signal tobe generated, and wherein the second probe oligonucleotides comprise: i)a second analyte specific region configured for detecting a secondallele at the polymorphic locus, ii) a label capable of generating adetectable signal or a cleavable portion configured to cause adetectable signal to be generated, wherein the plurality of second probeoligonucleotides are configured to occupy a probe hybridization site onthe amplified sequences at a different frequency than the plurality offirst probe oligonucleotides, and wherein the type of detectable signalfrom the first and second probe oligonucleotides is the same; c)measuring the strength of the detectable signal generated, therebydetermining the presence of the first allele, the second allele, or boththe first and second alleles in the viral target nucleic acid. Incertain embodiments, the polymorphic locus is a single nucleotidepolymorphism. In other embodiments, the polymorphic locus is a repeatsequence.

In some embodiments, the present invention provides methods fordetecting a viral target nucleic acid in a sample, comprising; a)incubating a sample suspected of containing a viral target nucleic acidwith a plurality of first and second probe oligonucleotides, a pluralityof upstream oligonucleotides, and a cleavage agent, wherein each of thefirst probe oligonucleotides comprise: i) a first analyte specificregion configured to hybridize to a probe hybridization site on theviral target nucleic acid, and ii) a first non-analyte specific regionconfigured to not hybridize to the viral target nucleic acid, whereinthe first non-analyte specific region is 5′ of the first analytespecific region, and wherein each of the second probe oligonucleotidescomprises i) a second analyte specific region configured to hybridize tothe probe hybridization site on the viral target nucleic acid, and ii) asecond non-analyte specific region configured to not hybridize to theviral target nucleic acid, wherein the second non-analyte specificregion is not identical to the first non-analyte specific region, andwherein the plurality of second probe oligonucleotides are configured tooccupy the probe hybridization site on the viral target nucleic acid ata different frequency than the plurality of first probeoligonucleotides; wherein the incubating is under conditions such thatinvasive cleavage structures are formed resulting in the cleavage ofboth the first and second probe oligonucleotides by the cleavage agentto generate: i) first non-viral target cleavage products comprising thefirst non-analyte specific region, and ii) second non-viral targetcleavage products comprising the second non-analyte specific region; andb) measuring hybridization of the first and the second probeoligonucleotides by detecting a signal generated by the first and secondnon-viral target cleavage products, thereby detecting the viral targetnucleic acid. In some embodiments, the amount of the viral target isdetected.

In certain embodiments, the present invention provides methods fordetecting a viral target nucleic acid in a sample, comprising; a)incubating a sample suspected of containing a viral target nucleic acidwith a plurality of first and second probe oligonucleotides, a pluralityof first upstream oligonucleotides, a plurality of second upstreamoligonucleotides, and a cleavage agent, wherein each of the first probeoligonucleotides comprise: i) a first analyte specific region configuredto hybridize to a first probe hybridization site on the viral targetnucleic acid, and ii) a first non-analyte specific region configured tonot hybridize to the viral target nucleic acid, wherein the firstnon-analyte specific region is 5′ of the first analyte specific region,and wherein each of the second probe oligonucleotides comprises i) asecond analyte specific region configured to hybridize to a second probehybridization site on the viral target nucleic acid, wherein the secondprobe hybridization site is not the same as the first probehybridization site, and ii) a second non-analyte specific regionconfigured to not hybridize to the viral target nucleic acid, whereinthe second non-analyte specific region is not identical to the firstnon-analyte specific region, and wherein the plurality of second probeoligonucleotides are present in at least 5-fold lower concentration thanthe first probe oligonucleotides; wherein the incubating is underconditions such that invasive cleavage structures are formed resultingin the cleavage of both the first and second probe oligonucleotides bythe cleavage agent to generate: i) first non-viral target cleavageproducts comprising the first non-analyte specific region, and ii)second non-viral target cleavage products comprising the secondnon-analyte specific region; and b) measuring hybridization of the firstand the second probe oligonucleotides over time by detecting a signalgenerated by the first and second non-viral target cleavage products,thereby measuring the amount of the viral target nucleic acid. In someembodiments, the amount of the viral target is detected.

In certain embodiments, the second probe oligonucleotides are present inat least a 10-fold, 100-fold, or 1000-fold, lower concentration than thefirst probe oligonucleotides. In further embodiments, the signalgenerated by the first and second non-viral target cleavage products isthe same. In other embodiments, the signal generated by the first andsecond non-viral target cleavage products is different. In someembodiments, the upstream oligonucleotides comprise a 5′ portion and a3′ portion, wherein the 5′ portion is configured to hybridize to aregion contiguous with the probe hybridization site on the viral targetnucleic acid, and wherein the 3′ portion is configured to not hybridizeto the viral target nucleic acid. In further embodiments, the methodsfurther comprise incubating the sample with first and second labeledsequences, wherein the first labeled sequence is configured to generatea first detectable signal when hybridized to the first non-viral targetcleavage product, and wherein the second labeled sequence is configuredto generate a second detectable signal when hybridized to the secondnon-viral target cleavage product. In particular embodiments, the firstand second detectable signals are the same. In additional embodiments,the first and second labeled sequences comprise FRET cassettes. In otherembodiments, the plurality of upstream oligonucleotides are generated inthe sample (e.g., by a polymerase). In some embodiments, the upstreamoligonucleotides are supplied pre-synthesized.

In certain embodiments, the present invention provides kits forquantitation of viral target nucleic acids in a sample, comprising: a) aplurality of first probe oligonucleotides, wherein each of the firstprobe oligonucleotides comprises a first analyte specific region,wherein the first probe oligonucleotides are un-labeled, or comprise alabel, b) a plurality of second probe oligonucleotides, wherein each ofthe second probe oligonucleotides comprises a second analyte specificregion, wherein the second probe oligonucleotides are un-labeled, orcomprise a label, wherein the plurality of second probe oligonucleotidesare configured to occupy a probe hybridization site on the viral targetnucleic acids at a different frequency than the plurality of first probeoligonucleotides; and c) reagents for performing an INVADER assay usingthe pluralities of the first and second probe oligonucleotides.

In certain embodiments, the present invention provides kits orcompositions comprising: i) a plurality of first oligonucleotides, andii) a plurality of second probe oligonucleotides, wherein the firstprobe oligonucleotides comprise a first 5′ region and a first 3′ region,and the second probe oligonucleotides comprises a second 5′ region and asecond 3′ region, wherein both of the first and second probeoligonucleotides will form an invasive cleavage structure in thepresence of the same upstream oligonucleotide and viral target sequence,and will both be cleaved by the same cleavage agent to form a first 5′region product and a second 5′ region product, wherein the second 5′region product is not identical to the first 5′ region product. In someembodiments, the kit or composition further comprises iii) first andsecond labeled sequences, wherein the first labeled sequence isconfigured to generate a first detectable signal when hybridized to thefirst 5′ region product, and wherein the second labeled sequence isconfigured to generate a second detectable signal when hybridized to thesecond 5′ region product. In some embodiments, the kits further comprisethe viral target sequence as a control.

In particular embodiments, the first and second probe oligonucleotidesare provides in a first vessel. In further embodiments, the, and kitfurther comprises a second vessel containing a polymerase and FENenzyme. In additional embodiments, the kit further comprises a thirdvessel containing a buffer.

In some embodiments, the first 3′ region and the second 3′ region havethe identical sequence. In other embodiments, the first 3′ region andthe second 3′ region do not have identical sequences. In particularembodiments, the second probe oligonucleotides are present in at least a5 fold lower concentration than the first probe oligonucleotides. Inother embodiments, the second probe oligonucleotides are present in atleast a 10 fold . . . 100-fold . . . 1000-fold . . . 10,000-fold . . .or 500,000 lower concentration than the first probe oligonucleotides. Insome embodiments, the first and second probe oligonucleotides areun-labeled.

In further embodiments, the kits or compositions further comprise athird probe oligonucleotide comprising a third 5′ region and a third 3′region, wherein the third probe oligonucleotide will not form aninvasive cleavage structure with the viral target and the upstreamoligonucleotide that is cleavable by the cleavage agent. In someembodiments, the first and second detectable signals are the same orthey are different.

In some embodiments, the present invention provides kits comprising i) aplurality of un-labeled first probe oligonucleotides and ii) a pluralityof un-labeled second probe oligonucleotides, wherein the first andsecond probe oligonucleotides comprises an analyte specific region,wherein the plurality of second probe oligonucleotides are configured tooccupy a probe hybridization site on a viral target nucleic acid at adifferent frequency than the plurality of first probe oligonucleotides.In further embodiments, the kits further comprise a polymerase and/or aFEN enzyme. In other embodiments, the kits further comprise a buffer.

In some embodiments, the present invention provides kits comprising; a)a first vessel comprising a plurality of first probe oligonucleotides(e.g., unlabeled) and a plurality of second probe oligonucleotides(e.g., unlabeled), wherein the first and second probe oligonucleotidescomprises an analyte specific region, wherein the plurality of secondprobe oligonucleotides are configured to occupy a probe hybridizationsite on a viral target nucleic acid at a different frequency than theplurality of first probe oligonucleotides; b) a second vessel comprisinga polymerase and/or a FEN enzyme, and c) a third vessel comprising abuffer. In certain embodiments, the kits further comprise d) a controlviral target sequence comprising the probe hybridization site.

In particular embodiments, the present invention provides kits andcompositions comprising; a) a plurality of first and second probeoligonucleotides, wherein the first probe oligonucleotides comprise afirst 5′ region and a first 3′ region, and the second probeoligonucleotides comprises a second 5′ region and a second 3′ region,wherein both of the first and second probe oligonucleotides will form aninvasive cleavage structure in the presence of the same upstreamoligonucleotide and viral target sequence, and will both be cleaved bythe same cleavage agent to form a first 5′ region product and a second5′ region product, wherein the second 5′ region product is identical tothe first 5′ region product, and wherein the first 3′ region is notidentical to the second 3′ region, and b) first labeled sequences,wherein the first labeled sequence is configured to generate adetectable signal when hybridized to the first or second 5′ regionproduct.

In some embodiments, the present invention provides methods fordetecting at least two different viral, type or subtypes, species in asingle reaction vessel comprising; a) providing a reaction vesselcontaining a sample, wherein the sample is suspected of containing firstand second viral target nucleic acids, wherein the first viral targetnucleic acids are from a viral species different from the second viraltarget nucleic acids; b) incubating the sample in the reaction vesselwith a plurality of first and second probes, a plurality of first andsecond reporter sequences, and a cleavage agent under conditions suchthat: i) the first probes hybridize to first viral target nucleic acidsand are cleaved by the cleavage agent thereby generating first 5′cleaved portions, and ii) the second probes hybridize to second viraltarget nucleic acids and are cleaved by the cleavage agent therebygenerating second 5′ cleaved portions; and c) detecting a signal fromthe sample in the reaction vessel, wherein the signal is from the firstand second reporter sequences and is generated by hybridization of thefirst 5′ cleaved portions to the first reporter sequences andhybridization of the second 5′ cleaved portions to the second reportersequences, thereby detecting the presence of both the first and secondtarget nucleic acids in the sample.

In certain embodiments, the present invention provides methods fordetecting at least two different viral species in a single reactionvessel comprising; a) providing a reaction vessel containing a sample,wherein the sample is suspected of containing first and second viraltarget nucleic acids, wherein the first viral target nucleic acids arefrom a viral species different from the second viral target nucleicacids; b) incubating the sample in the reaction vessel with a pluralityof first and second probes, a plurality of reporter sequences, and acleavage agent under conditions such that: i) the first probes hybridizeto first viral target nucleic acids and are cleaved by the cleavageagent thereby generating first 5′ cleaved portions, and ii) the secondprobes hybridize to second viral target nucleic acids and are cleaved bythe cleavage agent thereby generating second 5′ cleaved portions; and c)detecting a signal from the sample in the reaction vessel, wherein thesignal is from the reporter sequences and is generated by hybridizationof the first and second 5′ cleaved portions to the reporter sequencesthereby detecting the presence of both the first and second targetnucleic acids in the sample. In particular embodiments, the first andsecond 5′ cleaved portions are identical to each other.

In other embodiments, the first viral target nucleic acid comprises atleast a portion of a first viral genome (e.g., hepatitis C virus) or anamplified portion of the first viral genome. In certain embodiments, thesecond viral target nucleic acid comprises at least a portion of asecond viral genome or an amplified portion of the second viral genome.In particular embodiments, the first viral target nucleic acids compriseat least a portion of, or an amplified portion of, a RNA viral genomeand the second viral target nucleic acids comprise at least a portionof, or an amplified portion of, a DNA viral genome. In some embodiments,the first viral target nucleic acids comprise cDNA copies of a portionof a first viral genome. In other embodiments, the first or second viraltarget nucleic acids are amplified prior to step b). In someembodiments, the first and second viral target nucleic acids areamplified prior to step b). In additional embodiments, the signal isgenerated by cleavage of the reporter sequences.

In certain embodiments, the first and second probes are cleaved as partof an invasive cleavage reaction. In certain embodiments, the incubatingfurther comprises incubating the sample with first upstreamoligonucleotides configured to form invasive cleavage structures withthe first probes and the first viral target nucleic acids. In otherembodiments, the incubating further comprises incubating the sample withsecond upstream oligonucleotides configured to form invasive cleavagestructures with the second probes and the second target nucleic acids.

In particular embodiments, the reporter sequences comprise a dye and aquencher. In other embodiments, the first and second 5′ cleaved portionsare configured to not hybridize to the first and second viral targetnucleic acids. In some embodiments, the first viral target nucleic acidcomprises at least a portion of an HCV viral genome or an amplifiedportion of the HCV viral genome. In other embodiments, the second viraltarget nucleic acid comprises at least a portion of and HSV-1 viralgenome or an amplified portion of the HSV-1 viral genome.

In some embodiments, the present invention provides methods fordetecting the presence or absence of a target nucleic acid in a samplecomprising; a) incubating the sample with a stem oligonucleotide, anupstream oligonucleotide, a downstream probe, and a cleavage agent underconditions such that, if the target nucleic acid is present: i) a 3′target specific region of the stem oligonucleotide hybridizes to thetarget nucleic acid, and a stem region of the stem oligonucleotideremains available for hybridization to a portion of the upstreamoligonucleotide and the downstream probe, ii) a 5′ target specificregion of the upstream oligonucleotide hybridizes to the target nucleicacid, and a stem specific region of the upstream oligonucleotidehybridizes to a portion of the stem region of the stem oligonucleotide,iii) a 3′ region of the downstream probe hybridizes to a portion of thestem region of the stem oligonucleotide, and a 5′ region of thedownstream probe does not hybridize to the stem region of the stemoligonucleotide, wherein the downstream probe forms an invasive cleavagestructure with the upstream oligonucleotide and the stem region of thestem oligonucleotide, and iv) the cleavage agent cleaves the downstreamprobe in the invasive cleavage structure thereby generating a 5′ cleavedportion; and b) detecting the presence or absence of the target nucleicacid in the sample.

In certain embodiments, the detecting comprises determining if the 5′cleaved portion has been generated. In other embodiments, the incubatingfurther includes a reporter sequence, and the detecting the presence orabsence of the target nucleic acid comprises detecting a signal from thereporter sequence generated by hybridization of the 5′ cleaved portionto the reporter sequence. In particular embodiments, the hybridizationof the 5′ cleaved portion to the reporter sequence generates an invasivecleavage structure that is cleaved by the cleavage agent.

In certain embodiments, the target nucleic acid is composed of RNA andthe reporter sequence is composed of DNA. In further embodiments, thetarget nucleic acid comprises RNA. In other embodiments, the targetnucleic acid comprises viral RNA. In some embodiments, the upstreamoligonucleotide comprises a 3′ region configured to not hybridize to thestem region of the stem oligonucleotide. In further embodiments, theupstream oligonucleotide comprises a 3′ region configured to hybridizeto the stem region of the stem oligonucleotide. In other embodiments,the stem specific region of the upstream oligonucleotide is between 5and 12 bases in length (e.g., 5, 6, 7, 8, 9, 10, 11, or 12 bases inlength). In particular embodiments, the stem specific region of theupstream oligonucleotide is 8 or 9 bases in length.

In some embodiments, the 5′ target specific region of the upstreamoligonucleotide is partially or fully complementary to the targetnucleic acid. In other embodiments, the 5′ target specific region of theupstream oligonucleotide is between 20 and 60 bases in length (e.g., 20,30, 40, 50, or 60 bases). In additional embodiments, the 5′ targetspecific region of the upstream oligonucleotide has a Tm of about 20degrees Celsius (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25degrees Celsius).

In other embodiments, the 3′ target specific region of the stemoligonucleotide is partially or fully complementary to the targetnucleic acid. In certain embodiments, the 3′ target specific region ofthe stem oligonucleotide is between 15 and 50 bases in length (e.g., 15. . . 25 . . . 35 . . . or 50 bases in length). In some embodiments, thestem and upstream oligonucleotides are present in step a) at aconcentration of less than 10 nM. In other embodiments, the stem andupstream oligonucleotides are present in step a) at a concentration ofabout 100-300 pM (e.g., 100 pM . . . 150 pM . . . 200 pM . . . 250 pM .. . or 300 pM).

In certain embodiments, polymerization is employed to create at least aportion of one or more of the following sequences: the stemoligonucleotide, the upstream, or the downstream probe. In otherembodiments, the stem oligonucleotide is blocked on the 3′ end, the 5′end, or both the 3′ and the 5′ ends (e.g. with one or more2′-O-methylated bases). In further embodiments, the upstreamoligonucleotide is blocked on the 5′ end. (e.g. with one or more2′-O-methylated bases).

In some embodiments, the incubating further includes a polymerase, aprimer, and dNTPs, and wherein the incubating is under conditions suchthat the polymerase extends the 3′ end of the upstream oligonucleotideusing the stem region of the stem oligonucleotide as a template togenerate an extended upstream oligonucleotide that comprises an upstreamoligonucleotide extended region. In other embodiments, the methodsfurther comprise heating the sample in order to separate the extendedupstream oligonucleotide from the stem oligonucleotide and the targetnucleic acid. In particular embodiments, the methods further comprisecooling the sample under conditions such that the primer hybridizes toat least a portion of the upstream oligonucleotide extended region ofthe extended upstream oligonucleotide. In additional embodiments, themethods further comprise incubating the sample under conditions suchthat the primer is extended by the polymerase using the extendedupstream oligonucleotide as a template such that a stem ampliconsequence is generated. In particular embodiments, the methods furthercomprise heating the sample in order to separate the stem ampliconsequence from the extended upstream oligonucleotide. In otherembodiments, the methods further comprise incubating the sample with adownstream probe and upstream oligonucleotide such that an invasivecleavages structure is formed with the stem amplicon sequence, thedownstream probe, and the upstream oligonucleotide.

In some embodiments, the present invention provides methods of targetnucleic acid dependent amplification of a non-target sequence in asample comprising; a) incubating the sample with stem oligonucleotides,upstream oligonucleotides, primers, dNTPs, and a polymerase underconditions such that, if the target nucleic acid is present: i) a 3′target specific region of the stem oligonucleotides hybridizes to thetarget nucleic acid, and a stem region of the stem oligonucleotidesremains available for hybridization to the upstream oligonucleotides,ii) a 5′ target specific region of the upstream oligonucleotideshybridizes to the target nucleic acid, and a stem specific region of theupstream oligonucleotides hybridizes to a portion of the stem region ofthe stem oligonucleotides, and iii) the polymerase extends the 3′ end ofthe upstream oligonucleotides using the stem region of the stemoligonucleotides as a template to generate extended upstreamoligonucleotides that comprise an upstream oligonucleotide extendedregion; b) heating the sample in order to separate the extended upstreamoligonucleotides from the stem oligonucleotides and the target nucleicacid; c) cooling the sample under conditions such that the primershybridize to at least a portion of the upstream oligonucleotide extendedregion of the extended upstream oligonucleotides; and d) incubating thesample under conditions such that the primers are extended by thepolymerase using the extended upstream oligonucleotide as a templatesuch that stem amplicon sequences are generated.

In certain embodiments, the methods further comprise the step ofdetecting the presence or absence of the target nucleic acid in thesample by determining if stem amplicon sequences are generated. Infurther embodiments, the methods further comprise the step of performingone or more rounds of PCR using the stem amplicon sequences and theextended upstream oligonucleotides as templates, wherein the upstreamoligonucleotides prime polymerization from the stem amplicon sequences,and wherein the primers prime polymerization from the extended upstreamoligonucleotides. In additional embodiments, the methods furthercomprise a step after step f) of detecting the presence or absence ofthe target nucleic acid in the sample by detecting any accumulated PCRproducts. In particular embodiments, the methods further comprise a stepprior to step of heating the sample in order to separate the stemamplicon sequences from the extended upstream oligonucleotides. In someembodiments, the methods further comprise incubating the sample withdownstream probes and upstream oligonucleotides such that invasivecleavage structure are formed with the stem amplicon sequences, thedownstream probes, and the upstream oligonucleotides.

In particular embodiments, the present invention provides kits orcompositions (e.g., reaction mixtures) comprising; a) a stemoligonucleotide, b) an upstream oligonucleotide, wherein the upstreamoligonucleotide is configured to stably hybridize to the stemoligonucleotide only when both the stem and upstream oligonucleotidesare hybridized to a target sequence, and c) a downstream probe, whereinthe downstream probe comprises a 3′ region configured to hybridize to aportion of the stem oligonucleotide and a 5′ portion configured to nothybridize to the stem oligonucleotide, and wherein the downstream probeis configured to form an invasive cleavage structure with the upstreamand stem oligonucleotides in the presence of the target sequence. Incertain embodiments, the kits or compositions comprising a cleavageagent.

In some embodiments, the present invention provides kits andcompositions (e.g., reaction mixtures) comprising; a) a stemoligonucleotide, b) an upstream oligonucleotide, wherein the upstreamoligonucleotide is configured to stably hybridize to the stemoligonucleotide only when both the stem and upstream oligonucleotidesare hybridized to a target sequence, and c) a primer, wherein the primeris configured to hybridize to a region that is created by extending the3′ end of the upstream oligonucleotide with a polymerase and dNTPs whenthe stem and upstream oligonucleotides are hybridized together andhybridized to the target sequence. In other embodiments, the kits orcompositions further comprise a polymerase.

In certain embodiments, the present invention provides methods, kits,compositions, and devices for nucleic dispensing, where the nucleic acidcomprises a non-ionic detergent. In some embodiments, the presentinvention provides methods nucleic acid dispensing comprising; a)dispensing a nucleic acid mixture comprising a plurality ofoligonucleotides onto a first surface region (e.g., a well) such that afirst amount of the oligonucleotides are delivered to the first surfaceregion, wherein the dispensing is through a hydrophobic polymerdispensing component (e.g. pipette tip) attached to, or integral with, aliquid dispensing device, and wherein the nucleic acid mixture furthercomprises a non-ionic detergent; and b) dispensing the nucleic acidmixture onto at least 15 additional surface regions (e.g., wells, whichmay or may not be part of the same device or component, such as a plate)through the hydrophobic polymer dispensing component attached to theliquid dispensing device such that the amount of the oligonucleotidesdelivered to each of the at least 15 additional surface regions iswithin about 15 percent of the first amount.

In particular embodiments, the first and/or additional surface regionsare wells in the same plate. In other embodiments, the first and/oradditional surface regions are wells in separate plates. In someembodiments, the first and/or additional surface regions are processchamber in the same or different microfluidic device components. Inadditional embodiments, the first and/or additional surface regions atest tubes, beakers, flasks, or other surface where it is desired toplace a nucleic acid mixture.

In certain embodiments, the present invention provides liquid dispensingdevice comprising; a) a liquid holding reservoir, wherein the liquidholding reservoir contains a nucleic acid mixture comprising a pluralityof oligonucleotides and a non-ionic detergent, wherein the non-ionicdetergent makes up between about 0.005% and about 5.0% by volume of thenucleic acid mixture; b) at least one liquid dispensing channel operablylinked to the liquid holding reservoir, c) a hydrophobic polymerdispensing component (e.g. pipette tip) attached to the at least oneliquid dispensing channel.

In particular embodiments, the hydrophobic polymer dispensing componentcomprises a dispensing tip (e.g., NanoScreen or Beckman pipette tip). Incertain embodiments, the hydrophobic polymer dispensing component iscomposed of a material comprising polypropylene. In other embodiments,the hydrophobic polymer dispensing component is composed of a materialselected from the group consisting of: polytetrafluoroethylene,polyethylene, or polypropylene.

In some embodiments, the amount of the oligonucleotides delivered toeach of the additional surface regions is within about 10 percent of thefirst amount. In further embodiments, the amount of the oligonucleotidesdelivered to each of the additional surface regions is within about 7percent of the first amount. In particular embodiments, the first amountdelivered to the first surface region is the first contact of thehydrophobic polymer component with the nucleic acid mixture. In otherembodiments, the first amount delivered to the first surface region isnot the first contact of the hydrophobic polymer component with thenucleic acid mixture (e.g., the first amount is the tenth, or fifteenth,or thirty-first amount dispensed from the liquid dispensing devicethrough the particular hydrophobic polymer component).

In certain embodiments, the non-ionic detergent makes up between about0.005% and about 5.0% by volume of the nucleic acid mixture (e.g.,0.005% . . . 0.009% . . . 0.0015% . . . 0.09% . . . 0.55% . . . 1.0% . .. 2.5% . . . 4.0% . . . or 5%). In other embodiments, the non-ionicdetergent makes up between about 0.005% and about 0.25% by volume of thenucleic acid mixture. In further embodiments, the non-ionic detergentmakes up between about 0.005% and about 0.1% by volume of the nucleicacid mixture. In some embodiments, the non-ionic detergent makes upbetween about 0.005% and about 0.01% by volume of the nucleic acidmixture.

In certain embodiments, the at least 15 additional surface regions is at16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, or at least 24 additional surface regions. Inadditional embodiments, the at least 15 additional surface regions is atleast 25 additional surface regions (e.g., 25 . . . 35 . . . 45 . . . ,or 50). In particular embodiments, the at least 15 additional surfaceregions is at least 50 surface regions (e.g. 50 wells . . . 65 wells . .. or 74 wells). In additional embodiments, the at least 15 additionalsurface regions is at least 75 wells (e.g., 75 wells . . . 85 wells . .. 95 wells . . . or 99 wells). In further embodiments, the at least 15additional surface regions is at least 100 wells (e.g., 100 wells . . .125 wells . . . 150 wells . . . 175 . . . 200 wells . . . 250 wells . .. 500 well . . . 1000 wells . . . or 10,000 wells).

In certain embodiments, the oligonucleotides are configured to produce adetectable signal in the presence of a target nucleic acid sequence. Infurther embodiments, the oligonucleotides are configured to participatein a nucleic acid detection assay selected from the group consisting of:a TAQMAN assay, an INVADER assay, a sequencing assay, a polymerase chainreaction assay, a hybridization assay, a hybridization assay employing aprobe complementary to a mutation, a bead array assay, a primerextension assay, an enzyme mismatch cleavage assay, a branchedhybridization assay, a rolling circle replication assay, a NASBA assay,a molecular beacon assay, a cycling probe assay, a ligase chain reactionassay, or a sandwich hybridization assay.

In some embodiments, the liquid dispensing device comprises an automatedliquid dispensing device. In further embodiments, the liquid dispensingdevice comprises a hand-held liquid dispensing device. In otherembodiments, the hydrophobic polymer dispensing component is one of 5 .. . 12 . . . 24 . . . 96 . . . 192 . . . 384 . . . 768 . . . 1,500attached to the liquid dispensing device. In further embodiments, thevolume of liquid dispensed into the first well is between about 10 andabout 0.01 μL (e.g., 10 mL . . . 1 mL . . . 900 μL . . . 500 μL . . .250 μL . . . 100 μL . . . 50 μL . . . 20 μL . . . 10 μL . . . 5 μL . . .1 μL . . . 0.5 μL . . . 0.1 μL . . . or 0.01 Lμ). In some embodiments,the volume of liquid dispensed into each of the at least 15 additionalwells is within about 5% of the volume of liquid dispensed into thefirst well (e.g. within about 0.1%, 1%, 2%, 3%, 4%, or 5% of the volumedispensed into the first well).

In certain embodiments, the first surface region and the at least 15additional surface regions are formed in a microfluidic sampleprocessing device component. In some embodiments, the microfluidicsample processing device component is configured to be combined with oneor more additional components to generate a microfluidic sampleprocessing device. In other embodiments, the at least 15 additionalsurface regions are process chambers for a microfluidic sampleprocessing device. In further embodiments, the microfluidic sampleprocessing device comprises: i) the process chambers, ii) a plurality offeeder conduits, wherein each of the plurality of feeder conduits is influid communication with at least one of the process chambers, iii) amain conduit which is in fluid communication with the plurality offeeder conduits, and iv) a loading chamber which is in fluidcommunication with the main conduit.

In certain embodiments, the nucleic acid mixture delivered to the atleast 15 additional surface regions is dried down in the wells. Inparticular embodiments, the nucleic acid mixture contains a knownconcentration of a tracer dye. In further embodiments, the tracer dyecomprises a free label. In other embodiments, the tracer dye comprises afluorophore. In some embodiments, the tracer dye comprises a shortoligonucleotide linked to a label (e.g. an oligonucleotide between 5 and15 bases with a know sequence). In additional embodiments, the labelcomprises a fluorophore, chromophore, or radioactive label. In certainembodiments, the tracer dye comprises a mixture of both a free label andoligonucleotide linked label.

Other embodiments of the invention are described in the DetailedDescription of the Invention and the Examples.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “dynamic range” refers to the quantitativerange of usefulness in a detection assay (e.g., a nucleic acid detectionassay). For example, the dynamic range of a viral detection assay is therange between the smallest number of viral particles (e.g., copy number)and the largest number of viral particles that the assay can distinguishbetween.

As used herein, the terms “subject” and “patient” refer to any organismsincluding plants, microorganisms and animals (e.g., mammals such asdogs, cats, livestock, and humans).

The term “primer” refers to an oligonucleotide that is capable of actingas a point of initiation of synthesis when placed under conditions inwhich primer extension is initiated. An oligonucleotide “primer” mayoccur naturally, as in a purified restriction digest or may be producedsynthetically.

The term “cleavage structure” as used herein, refers to a structure thatis formed by the interaction of at least one probe oligonucleotide and atarget nucleic acid, forming a structure comprising a duplex, theresulting structure being cleavable by a cleavage means, including butnot limited to an enzyme. The cleavage structure is a substrate forspecific cleavage by the cleavage means in contrast to a nucleic acidmolecule that is a substrate for non-specific cleavage by agents such asphosphodiesterases, which cleave nucleic acid molecules without regardto secondary structure (i.e., no formation of a duplexed structure isrequired).

The term “invasive cleavage structure” as used herein refers to acleavage structure comprising i) a target nucleic acid, ii) an upstreamnucleic acid (e.g., an INVADER oligonucleotide), and iii) a downstreamnucleic acid (e.g., a probe), where the upstream and downstream nucleicacids anneal to contiguous regions of the target nucleic acid, and wherean overlap forms between the upstream nucleic acid and duplex formedbetween the downstream nucleic acid and the target nucleic acid. Anoverlap occurs where one or more bases from the upstream and downstreamnucleic acids occupy the same position with respect to a target nucleicacid base, whether or not the overlapping base(s) of the upstreamnucleic acid are complementary with the target nucleic acid, and whetheror not those bases are natural bases or non-natural bases. In someembodiments, the 3′ portion of the upstream nucleic acid that overlapswith the downstream duplex is a non-base chemical moiety such as anaromatic ring structure, e.g., as disclosed, for example, in U.S. Pat.No. 6,090,543, incorporated herein by reference in its entirety. In someembodiments, one or more of the nucleic acids may be attached to eachother, e.g., through a covalent linkage such as nucleic acid stem-loop,or through a non-nucleic acid chemical linkage (e.g., a multi-carbonchain).

The term “cleavage means” or “cleavage agent” as used herein refers toany means that is capable of cleaving a cleavage structure, includingbut not limited to enzymes. “Structure-specific nucleases” or“structure-specific enzymes” are enzymes that recognize specificsecondary structures in a nucleic molecule and cleave these structures.The cleavage means of the invention cleave a nucleic acid molecule inresponse to the formation of cleavage structures; it is not necessarythat the cleavage means cleave the cleavage structure at any particularlocation within the cleavage structure.

The cleavage means may include nuclease activity provided from a varietyof sources including the CLEAVASE enzymes, the FEN-1 endonucleases(including RAD2 and XPG proteins), Taq DNA polymerase and E. coli DNApolymerase I. The cleavage means may include enzymes having 5′ nucleaseactivity (e.g., Taq DNA polymerase (DNAP), E. coli DNA polymerase I).The cleavage means may also include modified DNA polymerases having 5′nuclease activity but lacking synthetic activity. Examples of cleavagemeans suitable for use in the method and kits of the present inventionare provided in U.S. Pat. Nos. 5,614,402; 5,795,763; 5,843,669; PCTAppln. Nos WO 98/23774; WO 02/070755A2; and WO0190337A2, each of whichis herein incorporated by reference it its entirety.

The term “thermostable” when used in reference to an enzyme, such as a5′ nuclease, indicates that the enzyme is functional or active (i.e.,can perform catalysis) at an elevated temperature, i.e., at about 55° C.or higher. In some embodiments the enzyme is functional or active at anelevated temperature of 65° C. or higher (e.g., 75° C., 85° C., 95° C.,etc.).

The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038[1972]). Other nucleic acid will not be replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (Chamberlin etal., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzymewill not ligate the two oligonucleotides or polynucleotides, where thereis a mismatch between the oligonucleotide or polynucleotide substrateand the template at the ligation junction (D. Y. Wu and R. B. Wallace,Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue oftheir ability to function at high temperature, are found to display highspecificity for the sequences bounded and thus defined by the primers;the high temperature results in thermodynamic conditions that favorprimer hybridization with the target sequences and not hybridizationwith non-target sequences (H. A. Erlich (ed.), PCR Technology, StocktonPress [1989]).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target.”In contrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

The term “analyte specific region” as used in reference to anoligonucleotide, such as a probe oligonucleotide or an INVADERoligonucleotide, refers to a region of an oligonucleotide selected tohybridize to a specific sequence in a target nucleic acid or set oftarget nucleic acids. In some embodiments, an analyte specific regionmay be completely complementary to the segment of a target nucleic acidto which it hybridizes, while in other embodiments, an analyte specificregion may comprise one or more mismatches to the segment of a targetnucleic acid to which it hybridizes. In yet other embodiments, ananalyte specific region may comprise one or more base analogs, e.g.,compounds that have altered hydrogen bonding, or that do not hydrogenbond, to the bases in the target strand. In some embodiments, the entiresequence of an oligonucleotide is an analyte specific region, while inother embodiments an oligonucleotide comprises an analyte specificregion and one or more regions not complementary the target sequence(e.g., non-complementary flap regions).

The term “frequency” as used herein in reference to hybridization ofnucleic acids refers to the probability that one particular nucleic acid(e.g., a probe oligonucleotide) will be base-paired to a complementarynucleic acid (e.g., a target nucleic acid) under particularhybridization conditions. The frequency of hybridization is influencedby many factors, including but not limited to the probability with whichthe complementary sequences will form a duplex under particularconditions (e.g., likelihood of encounter and of successful duplexformation) and the stability of the duplex, once formed. Reactionconditions that increase the likelihood of initial duplex formationbetween a probe and a target (e.g., increased concentration of one orboth nucleic acids, absence of competitors such as other nucleic acidswith sequences that can compete with a probe for binding to the target,or that can bind to the probe) can be said to increase the frequency ofhybridization of between the probe and target (i.e., increase thefrequency with which the probe oligonucleotide will occupy, or hybridizeto, the complementary target strand). Similarly, reaction conditions andprobe features that increase the stability of a hybrid between anoligonucleotide and another nucleic acid strand (or that slowdisassociation of the strands, e.g., reduced reaction temperature,increased salt or divalent cation conditions, increased length ofcomplementary regions, fewer mismatches, use of charged moietiesfavoring hybridization) can also be said to increase the frequency ofhybridization of between the probe and target. Conversely, reactionconditions and probe features that decrease the likelihood ofhybridization (e.g., reduction in concentration of one or both nucleicacids, the presence of a competitor or other additive that reduces theeffective concentration of a probe or target strand) or that reduce thestability and/or life time of hybrids that are formed (e.g., increasedreaction temperature, decreased salt or divalent cation conditions,decreased length of complementary regions, more mismatches, use ofcharged moieties disfavoring hybridization) are said to decrease thefrequency of hybridization or occupation.

As used herein, the term “target,” refers to a nucleic acid sequence orstructure to be detected or characterized. Thus, the “target” is soughtto be sorted out from other nucleic acid sequences. A “segment” isdefined as a region of nucleic acid within the target sequence.

The term “substantially single-stranded” when used in reference to anucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

As used herein, the phrase “non-amplified oligonucleotide detectionassay” refers to a detection assay configured to detect the presence orabsence of a particular target sequence (e.g. genomic DNA or viral DNAor RNA) that has not been amplified (e.g. by PCR), without creatingcopies of the target sequence. A “non-amplified oligonucleotidedetection assay” may, for example, amplify a signal used to indicate thepresence or absence of a particular polymorphism in a target sequence,so long as the target sequence is not copied.

The term “liberating” as used herein refers to the release of a nucleicacid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of, for example, a 5′ nuclease such thatthe released fragment is no longer covalently attached to the remainderof the oligonucleotide.

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi, and ciliates.

The term “microbial gene sequences” refers to gene sequences derivedfrom a microorganism.

The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery).

The term “multi-drug resistant” or multiple-drug resistant” refers to amicroorganism that is resistant to more than one of the antibiotics orantimicrobial agents used in the treatment of said microorganism.

The term “source of target nucleic acid” refers to any sample thatcontains nucleic acids (RNA or DNA). Particularly preferred sources oftarget nucleic acids are biological samples including, but not limitedto blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph,sputum and semen.

A sample “suspected of containing” a first and a second target nucleicacid may contain either, both or neither target nucleic acid molecule.

The term “reactant” is used herein in its broadest sense. The reactantcan comprise, for example, an enzymatic reactant, a chemical reactant orlight (e.g., ultraviolet light, particularly short wavelengthultraviolet light is known to break oligonucleotide chains). Any agentcapable of reacting with an oligonucleotide to either shorten (i.e.,cleave) or elongate the oligonucleotide is encompassed within the term“reactant.”

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid (e.g., 4, 5, 6, . . . , n−1).

The term “continuous strand of nucleic acid” as used herein is means astrand of nucleic acid that has a continuous, covalently linked,backbone structure, without nicks or other disruptions. The dispositionof the base portion of each nucleotide, whether base-paired,single-stranded or mismatched, is not an element in the definition of acontinuous strand. The backbone of the continuous strand is not limitedto the ribose-phosphate or deoxyribose-phosphate compositions that arefound in naturally occurring, unmodified nucleic acids. A nucleic acidof the present invention may comprise modifications in the structure ofthe backbone, including but not limited to phosphorothioate residues,phosphonate residues, 2′ substituted ribose residues (e.g., 2′-O-methylribose) and alternative sugar (e.g., arabinose) containing residues.

The term “continuous duplex” as used herein refers to a region of doublestranded nucleic acid in which there is no disruption in the progressionof basepairs within the duplex (i.e., the base pairs along the duplexare not distorted to accommodate a gap, bulge or mismatch with theconfines of the region of continuous duplex). As used herein the termrefers only to the arrangement of the basepairs within the duplex,without implication of continuity in the backbone portion of the nucleicacid strand. Duplex nucleic acids with uninterrupted basepairing, butwith nicks in one or both strands are within the definition of acontinuous duplex.

The term “duplex” refers to the state of nucleic acids in which the baseportions of the nucleotides on one strand are bound through hydrogenbonding the their complementary bases arrayed on a second strand. Thecondition of being in a duplex form reflects on the state of the basesof a nucleic acid. By virtue of base pairing, the strands of nucleicacid also generally assume the tertiary structure of a double helix,having a major and a minor groove. The assumption of the helical form isimplicit in the act of becoming duplexed.

The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

As used herein, the term “sample” is used in its broadest sense. Forexample, in some embodiments, it is meant to include a specimen orculture (e.g., microbiological culture), whereas in other embodiments,it is meant to include both biological and environmental samples (e.g.,suspected of comprising a target sequence, gene or template). In someembodiments, a sample may include a specimen of synthetic origin.

The present invention is not limited by the type of biological sampleused or analyzed. The present invention is useful with a variety ofbiological samples including, but are not limited to, tissue (e.g.,organ (e.g., heart, liver, brain, lung, stomach, intestine, spleen,kidney, pancreas, and reproductive (e.g., ovaries) organs), glandular,skin, and muscle tissue), cell (e.g., blood cell (e.g., lymphocyte orerythrocyte), muscle cell, tumor cell, and skin cell), gas, bodily fluid(e.g., blood or portion thereof, serum, plasma, urine, semen, saliva,etc), or solid (e.g., stool) samples obtained from a human (e.g., adult,infant, or embryo) or animal (e.g., cattle, poultry, mouse, rat, dog,pig, cat, horse, and the like). In some embodiments, biological samplesmay be solid food and/or feed products and/or ingredients such as dairyitems, vegetables, meat and meat by-products, and waste. Biologicalsamples may be obtained from all of the various families of domesticanimals, as well as feral or wild animals, including, but not limitedto, such animals as ungulates, bear, fish, lagamorphs, rodents, etc.

Biological samples also include biopsies and tissue sections (e.g.,biopsy or section of tumor, growth, rash, infection, orparaffin-embedded sections), medical or hospital samples (e.g.,including, but not limited to, blood samples, saliva, buccal swab,cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum,vomitus, bile, semen, oocytes, cervical cells, amniotic fluid, urine,stool, hair and sweat), laboratory samples (e.g., subcellularfractions), and forensic samples (e.g., blood or tissue (e.g., spatteror residue), hair and skin cells containing nucleic acids), andarcheological samples (e.g., fossilized organisms, tissue, or cells).

Environmental samples include, but are not limited to, environmentalmaterial such as surface matter, soil, water (e.g., freshwater orseawater), algae, lichens, geological samples, air containing materialscontaining nucleic acids, crystals, and industrial samples, as well assamples obtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items.

Other types of biological samples include bacteria (e.g., Actinobacteria(e.g., Actinomyces, Arthrobacter, Corynebacterium (e.g., C.diphtheriae)), Mycobacterium (e.g., M. tuberculosis and M. leprae),Propionibacterium (e.g., P. acnes), Streptomyces, Chlamydiae (e.g., C.trachomatis and C. pneumoniae), Cyanobacteria, Deinococcus (e.g.,Thermus (e.g., T. aquaticus)), Firmicutes (e.g., Bacilli (e.g., B.anthracis, B. cereus, B. thuringiensis, and B. subtilis)), Listeria(e.g., L. monocytogenes), Staphylococcus (e.g., S. aureus, S.epidermidis, and S. haemolyticus), Fusobacteria, Proteobacteria (e.g.,Rickettsiales, Sphingomonadales, Bordetella (e.g., B. pertussis),Neisserisales (e.g., N. gonorrhoeae and N. meningitidis),Enterobacteriales (e.g., Escherichia (e.g., E. coli), Klebsiella,Plesiomonas, Proteus, Salmonella, Shigella, and Yersinia),Legionellales, Pasteurellales (e.g., Haemophilus influenzae),Pseudomonas, Vibrio (e.g., V. cholerae and V. vulnificus),Campylobacterales (e.g., Campylobacteria (e.g., C. jejuni), andHelicobacter (e.g., H. pylori)), and Spirochaetes (e.g., Leptospira, B.bergdorferi, and T. pallidum)); Archaea (e.g., Halobacteria andMethanobacteria); Eucarya (e.g., Animalia (e.g., Annelidia, Arthropoda(e.g., Chelicerata, Myriapoda, Insecta, and Crustacea), Mollusca,Nematoda, (e.g., C. elegans, and T. spiralis) and Chordata (e.g.,Actinopterygii, Amphibia, Aves, Chondrichthyes, Reptilia, and Mammalia(e.g., Primates, Rodentia, Lagomorpha, and Carnivora)))); Fungi (e.g.,Dermatophytes, Fusarium, Penicillum, and Saccharomyces); Plantae (e.g.,Magnoliophyta (e.g., Magnoliopsida and Liliopsida)), and Protista (e.g.,Apicomplexa (e.g., Cryptosporidium, Plasmodium (e.g., P. falciparum, andToxoplasma), and Metamonada (e.g., G. lambia))); and Viruses (e.g.,dsDNA viruses (e.g., Bacteriophage, Adenoviridae, Herpesviridiae,Papillomaviridae, Polyomaviridae, and Poxyiridae), ssDNA virues (e.g.,Parvoviridae), dsRNA viruses (including Reoviridae), (+)ssRNA viruses(e.g., Coronaviridae, Astroviridae, Bromoviridae, Comoviridae,Flaviviridae, Picornaviridae, and Togaviridae), (−) ssRNA viruses (e.g.,Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae,and Orthomyxovirdiae), ssRNA-reverse transcribing viruses (e.g.,Retroviridae), and dsDNA-reverse transcribing viruses (e.g.,Hepadnaviridae and Caulomoviridae)).

Sample may be prepared by any desired or suitable method. In someembodiments, nucleic acids are analyzed directly from bodily fluids orother samples using the methods described in U.S. Pat. Pub. Serial No.20050186588, herein incorporated by reference in its entirety.

The above described examples are not, however, to be construed aslimiting the sample (e.g., suspected of comprising a target sequence,gene or template (e.g., the presence or absence of which can bedetermined using the compositions and methods of the present invention))types applicable to the present invention.

The terms “nucleic acid sequence” and “nucleic acid molecule” as usedherein refer to an oligonucleotide, nucleotide or polynucleotide, andfragments or portions thereof. The terms encompasses sequences thatinclude any of the known base analogs of DNA and RNA including, but notlimited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

A nucleic acid sequence or molecule may be DNA or RNA, of either genomicor synthetic origin, that may be single or double stranded, andrepresent the sense or antisense strand. Thus, nucleic acid sequence maybe dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, dsDNA made into ssDNA (e.g.,through melting, denaturing, helicases, etc.), A-, B-, or Z-DNA,triple-stranded DNA, RNA, ssRNA, dsRNA, mixed ss and dsRNA, dsRNA madeinto ssRNA (e.g., via melting, denaturing, helicases, etc.), messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA,snRNA, or protein nucleic acid (PNA).

The present invention is not limited by the type or source of nucleicacid (e.g., sequence or molecule (e.g. target sequence and/oroligonucleotide)) utilized. For example, the nucleic acid sequence maybe amplified or created sequence (e.g., amplification or creation ofnucleic acid sequence via synthesis (e.g., polymerization (e.g., primerextension (e.g., RNA-DNA hybrid primer technology)) and reversetranscription (e.g., of RNA into DNA)) and/or amplification (e.g.,polymerase chain reaction (PCR), rolling circle amplification (RCA),nucleic acid sequence based amplification (NASBA), transcriptionmediated amplification (TMA), ligase chain reaction (LCR), cycling probetechnology, Q-beta replicase, strand displacement amplification (SDA),branched-DNA signal amplification (bDNA), hybrid capture, and helicasedependent amplification).

The terms “nucleotide” and “base” are used interchangeably when used inreference to a nucleic acid sequence, unless indicated otherwise herein.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides including, but not limited to,analogs that have altered stacking interactions such as 7-deaza purines(i.e., 7-deaza-dATP and 7-deaza-dGTP); base analogs with alternativehydrogen bonding configurations (e.g., Iso-C and Iso-G and othernon-standard base pairs described in U.S. Pat. No. 6,001,983, hereinincorporated by reference in its entirety); non-hydrogen bonding analogs(e.g., non-polar, aromatic nucleoside analogs such as2,4-difluorotoluene, described by B. A. Schweitzer and E. T. Kool, J.Org. Chem., 1994, 59, 7238-7242; B. A. Schweitzer and E. T. Kool, J. Am.Chem. Soc., 1995, 117, 1863-1872, each of which is herein incorporate byreference in its entirety); “universal” bases such as 5-nitroindole and3-nitropyrrole; and universal purines and pyrimidines (e.g., “K” and “P”nucleotides, respectively; See, e.g., P. Kong, et al., Nucleic AcidsRes., 1989, 17, 10373-10383; P. Kong et al., Nucleic Acids Res., 1992,20, 5149-5152). Still other nucleotide analogs include modified forms ofdeoxyribonucleotides as well as ribonucleotides. Variousoligonucleotides of the present invention (e.g., a primary probe orINVADER oligo) may contain nucleotide analogs.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more nucleotides (e.g., deoxyribonucleotides orribonucleotides), preferably at least 5 nucleotides, more preferably atleast about 10-15 nucleotides and more preferably at least about 15 to30 nucleotides, or longer (e.g., oligonucleotides are typically lessthan 200 residues long (e.g., between 15 and 100 nucleotides), however,as used herein, the term is also intended to encompass longerpolynucleotide chains). The exact size will depend on many factors,which in turn depend on the ultimate function or use of theoligonucleotide. Oligonucleotides are often referred to by their length.For example a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes. Oligonucleotides may be generated inany manner, including chemical synthesis, DNA replication, reversetranscription, PCR, or a combination thereof. In some embodiments,oligonucleotides that form invasive cleavage structures are generated ina reaction (e.g., by extension of a primer in an enzymatic extensionreaction).

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide. Similarly, when two overlappingoligonucleotides are hybridized to the same linear complementary nucleicacid sequence, with the first oligonucleotide positioned such that its5′ end is upstream of the 5′ end of the second oligonucleotide, and the3′ end of the first oligonucleotide is upstream of the 3′ end of thesecond oligonucleotide, the first oligonucleotide may be called the“upstream” oligonucleotide and the second oligonucleotide may be calledthe “downstream” oligonucleotide.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (e.g., a sequence of two or morenucleotides (e.g., an oligonucleotide or a target nucleic acid)) relatedby the base-pairing rules. For example, the sequence “5′-A-G-T-3′,” iscomplementary to the sequence “3′-T-C-A-5′.” Complementarity may be“partial,” in which only some of the nucleic acid bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acid bases. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon the association of two ormore nucleic acid strands. Either term may also be used in reference toindividual nucleotides, especially within the context ofpolynucleotides. For example, a particular nucleotide within anoligonucleotide may be noted for its complementarity, or lack thereof,to a nucleotide within another nucleic acid sequence (e.g., a targetsequence), in contrast or comparison to the complementarity between therest of the oligonucleotide and the nucleic acid sequence.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallyhomologous sequence is one that is less than 100% identical to anothersequence. A partially complementary sequence that is “substantiallyhomologous” is a nucleic acid molecule that at least partially inhibitsa completely complementary nucleic acid molecule from hybridizing to atarget nucleic acid. The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (e.g., Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (e.g., the hybridization) of a completely homologous nucleicacid molecule to a target under conditions of low stringency. This isnot to say that conditions of low stringency are such that non-specificbinding is permitted (e.g., the low stringency conditions may be suchthat the binding of two sequences to one another be a specific (e.g.,selective) interaction). The absence of non-specific binding may betested by the use of a second target that is substantiallynon-complementary (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(e.g., is complementary to) the single-stranded nucleic acid sequenceunder conditions of low stringency as described above.

The terms “target nucleic acid” and “target sequence,” when used inreference to an invasive cleavage reaction, refer to a nucleic acidmolecule containing a sequence that has at least partial complementaritywith at least a first nucleic acid molecule (e.g. probe oligonucleotide)and may also have at least partial complementarity with a second nucleicacid molecule (e.g. INVADER oligonucleotide). Generally, the targetnucleic acid (e.g., present within, isolated from, enriched from, oramplified from or within a sample (e.g., a biological or environmentalsample)) is located within a target region and is identifiable via thesuccessful formation of an invasive cleavage structure in combinationwith a first and second nucleic acid molecule (e.g., probeoligonucleotide and INVADER oligonucleotide) that is cleavable by acleavage agent. Target nucleic acids from an organism are not limited togenomic DNA and RNA. Target nucleic acids from an organism may compriseany nucleic acid species, including but not limited to genomic DNAs andRNAs, messenger RNAs, structural RNAs, ribosomal and tRNAs, and smallRNAs such as snRNAs, siRNAs and microRNAs miRNAs). See, e.g., co-pendingU.S. patent application Ser. No. 10/740,256, filed Dec. 18, 2003, whichis incorporated herein by reference in its entirety.

As used herein, the term “probe oligonucleotide,” when used in referenceto an invasive cleavage reaction, refers to an oligonucleotide thatinteracts with a target nucleic acid to form a cleavage structure in thepresence or absence of an INVADER oligonucleotide. When annealed to thetarget nucleic acid, the probe oligonucleotide and target form acleavage structure and cleavage occurs within the probe oligonucleotide.

The term “INVADER oligonucleotide” refers to an oligonucleotide thathybridizes to a target nucleic acid at a location near the region ofhybridization between a probe and the target nucleic acid, wherein theINVADER oligonucleotide comprises a portion (e.g., a chemical moiety, ornucleotide—whether complementary to that target or not) that overlapswith the region of hybridization between the probe and target. In someembodiments, the INVADER oligonucleotide contains sequences at its 3′end that are substantially the same as sequences located at the 5′ endof a probe oligonucleotide.

The term “cassette,” when used in reference to an invasive cleavagereaction, as used herein refers to an oligonucleotide or combination ofoligonucleotides configured to generate a detectable signal in responseto cleavage of a probe oligonucleotide in an INVADER assay. In preferredembodiments, the cassette hybridizes to an cleavage product fromcleavage of the probe oligonucleotide to form a second invasive cleavagestructure, such that the cassette can then be cleaved.

In some embodiments, the cassette is a single oligonucleotide comprisinga hairpin portion (i.e., a region wherein one portion of the cassetteoligonucleotide hybridizes to a second portion of the sameoligonucleotide under reaction conditions, to form a duplex). In otherembodiments, a cassette comprises at least two oligonucleotidescomprising complementary portions that can form a duplex under reactionconditions. In preferred embodiments, the cassette comprises a label. Inparticularly preferred embodiments, the cassette comprises labeledmoieties that produce a fluorescence resonance energy transfer (FRET)effect.

An oligonucleotide is said to be present in “excess” relative to anotheroligonucleotide (or target nucleic acid sequence) if thatoligonucleotide is present at a higher molar concentration than theother oligonucleotide (or target nucleic acid sequence). When anoligonucleotide such as a probe oligonucleotide is present in a cleavagereaction in excess relative to the concentration of the complementarytarget nucleic acid sequence, the reaction may be used to indicate theamount of the target nucleic acid present. Typically, when present inexcess, the probe oligonucleotide will be present in at least a 100-foldmolar excess; typically at least 1 pmole of each probe oligonucleotidewould be used when the target nucleic acid sequence was present at about10 fmoles or less.

As used herein, the term “gene” refers to a nucleic acid (e.g., DNA)sequence that comprises coding sequences necessary for the production ofa polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (e.g., hnRNA); intronsmay contain regulatory elements (e.g., enhancers). Introns are removedor “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species (e.g.,a viral or bacterial gene present within a human host (e.g.,extrachromosomally or integrated into the host's DNA)). A heterologousgene also includes a gene native to an organism that has been altered insome way (e.g., mutated, added in multiple copies, linked to non-nativeregulatory sequences, etc). In some embodiments, a heterologous gene canbe distinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (e.g., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (e.g., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (e.g., these flanking sequences canbe located 5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and or functional properties (e.g., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally occurring mutants can be isolated (e.g.,identified by the fact that they have altered characteristics (e.g.,altered nucleic acid sequences) when compared to the wild-type gene orgene product).

The term “isolated” when used in relation to a nucleic acid (e.g., “anisolated oligonucleotide” or “isolated polynucleotide” or “an isolatednucleic acid sequence”) refers to a nucleic acid sequence that isseparated from at least one component or contaminant with which it isordinarily associated in its natural source. Thus, an isolated nucleicacid is present in a form or setting that is different from that inwhich it is found in nature. In contrast, non-isolated nucleic acids arenucleic acids such as DNA and RNA found in the state they exist innature. For example, a given DNA sequence (e.g., a gene) is found on thehost cell chromosome in proximity to neighboring genes; RNA sequences,such as a specific mRNA sequence encoding a specific protein, are foundin the cell as a mixture with numerous other mRNAs that encode amultitude of proteins. However, isolated nucleic acid encoding a givenprotein includes, by way of example, such nucleic acid in cellsordinarily expressing the given protein where the nucleic acid is in achromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (e.g., theoligonucleotide or polynucleotide may be single-stranded), but maycontain both the sense and anti-sense strands (e.g., the oligonucleotideor polynucleotide may be double-stranded).

As used herein, the terms “purified” or “to purify” when used inreference to a sample (e.g., a molecule (e.g., a nucleic acid or aminoacid sequence)) refers to removal (e.g., isolation and/or separation) ofthe sample from its natural environment. The term “substantiallypurified” refers to a sample (e.g., molecule (e.g. a nucleic acid oramino acid sequence) that has been removed (e.g., isolated and/orpurified) from its natural environment and is at least 60% free,preferably 75% free, or most preferably 90% or more free from othercomponents with which it is naturally associated. An “isolatedpolynucleotide” or “isolated oligonucleotide” may therefore besubstantially purified if it is rendered free (e.g., 60%, 75% or morepreferably 90% or more) from other components with which it is naturallyassociated.

The present invention is not limited to any particular means ofpurification (e.g., to generate purified or substantially purifiedmolecules (e.g., nucleic acid sequences)). Indeed, a variety ofpurification techniques may be utilized including, but not limited to,centrifugation (e.g., isopycnic, rate-zonal, gradient, and differentialcentrifugation), electrophoresis (e.g., gel and capillaryelectrophoresis), gel filtration, matrix capture, charge capture, masscapture, antibody capture, magnetic separation, flow cytometry, andsequence-specific hybridization array capture.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (e.g., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theT_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (See, e.g., Young and Anderson,(1985) in Nucleic Acid Hybridisation: A Practical Approach (Hames &Higgins, Eds.) pp 47-71, IRL Press, Oxford). Other computations forcalculating T_(m) are known in the art and take structural andenvironmental, as well as sequence characteristics into account (See,e.g., Allawi, H. T. and SantaLucia, J., Jr. Biochemistry 36, 10581-94(1997)).

As used herein, the term “INVADER assay reagents” refers to one or morereagents for detecting target sequences, said reagents comprisingnucleic acid molecules capable of forming an invasive cleavage structurein the presence of the target sequence. In some embodiments, the INVADERassay reagents further comprise an agent for detecting the presence ofan invasive cleavage structure (e.g., a cleavage agent). In someembodiments, the nucleic acid molecules comprise first and secondoligonucleotides, said first oligonucleotide comprising a 5′ portioncomplementary to a first region of the target nucleic acid and saidsecond oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′portion complementary to a second region of the target nucleic aciddownstream of and contiguous to the first portion. In some embodiments,the 3′ portion of the second oligonucleotide comprises a 3′ terminalnucleotide not complementary to the target nucleic acid. In preferredembodiments, the 3′ portion of the second oligonucleotide consists of asingle nucleotide not complementary to the target nucleic acid. INVADERassay reagents may be found, for example, in U.S. Pat. Nos. 5,846,717;5,985,557; 5,994,069; 6,001,567; 6,913,881; and 6,090,543, WO 97/27214,WO 98/42873, U.S. Pat. Publ. Nos. 20050014163, 20050074788, 2005016596,20050186588, 20040203035, 20040018489, and 20050164177; U.S. patentapplication Ser. No. 11/266,723; and Lyamichev et al., Nat. Biotech.,17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), each of which isherein incorporated by reference in its entirety for all purposes.

In some embodiments, INVADER assay reagents are configured to detect atarget nucleic acid sequence comprising first and second non-contiguoussingle-stranded regions separated by an intervening region comprising adouble-stranded region. In certain embodiments, the INVADER assayreagents comprise a bridging oligonucleotide capable of binding to saidfirst and second non-contiguous single-stranded regions of a targetnucleic acid sequence. In particularly preferred embodiments, either orboth of said first and/or said second oligonucleotides of said INVADERassay reagents are bridging oligonucleotides.

In some embodiments, the INVADER assay reagents further comprise a solidsupport. For example, in some embodiments, the one or moreoligonucleotides of the assay reagents (e.g., first and/or secondoligonucleotide, whether bridging or non-bridging) is attached to saidsolid support. The one or more oligonucleotides of the assay reagentsmay be linked to the solid support directly or indirectly (e.g., via aspacer molecule (e.g., an oligonucleotide)). Exemplary solid phaseinvasive cleavage reactions are described in U.S. Pat. Pub. Nos.20050164177 and 20030143585, herein incorporated by reference in theirentireties.

As used herein, a “solid support” is any material that maintains itsshape under assay conditions, and that can be separated from a liquidphase. The present invention is not limited by the type of solid supportutilized. Indeed, a variety of solid supports are contemplated to beuseful in the present invention including, but not limited to, a bead,planar surface, controlled pore glass (CPG), a wafer, glass, silicon,plastic, paramagnetic bead, magnetic bead, latex bead, superparamagneticbead, plurality of beads, microfluidic chip, a silicon chip, amicroscope slide, a microplate well, a silica gel, a polymeric membrane,a particle, a derivatized plastic film, a glass bead, cotton, a plasticbead, an alumina gel, a polysaccharide, polyvinylchloride,polypropylene, polyethylene, nylon, Sepharose, poly(acrylate),polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose,dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin,nitrocellulose, diazocellulose or starch, polymeric microparticle,polymeric membrane, polymeric gel, glass slide, styrene, multi-wellplate, column, microarray, latex, hydrogel, porous 3D hydrophilicpolymer matrix (e.g., HYDROGEL, Packard Instrument Company, Meriden,Conn.), fiber optic bundles and beads (e.g., BEADARRAY (Illumina, SanDiego, Calif.), described in U.S. Pat. App. 20050164177), smallparticles, membranes, frits, slides, micromachined chips,alkanethiol-gold layers, non-porous surfaces, addressable arrays, andpolynucleotide-immobilizing media (e.g., described in U.S. Pat. App.20050191660). In some embodiments, the solid support is coated with abinding layer or material (e.g., gold or streptavidin).

In some embodiments, the INVADER assay reagents further comprise abuffer solution. In some preferred embodiments, the buffer solutioncomprises a source of divalent cations (e.g., Mn²⁺ and/or Mg²⁺ ions).Individual ingredients (e.g., oligonucleotides, enzymes, buffers, targetnucleic acids) that collectively make up INVADER assay reagents aretermed “INVADER assay reagent components.”

In some embodiments, the INVADER assay reagents further comprise a thirdoligonucleotide complementary to a third portion of the target nucleicacid upstream of the first portion of the first target nucleic acid(e.g., a stacker oligonucleotides). In yet other embodiments, theINVADER assay reagents further comprise a target nucleic acid. In someembodiments, the INVADER assay reagents further comprise a second targetnucleic acid. In yet other embodiments, the INVADER assay reagentsfurther comprise a third oligonucleotide comprising a 5′ portioncomplementary to a first region of the second target nucleic acid. Insome specific embodiments, the 3′ portion of the third oligonucleotideis covalently linked to the second target nucleic acid. In otherspecific embodiments, the second target nucleic acid further comprises a5′ portion, wherein the 5′ portion of the second target nucleic acid isthe third oligonucleotide. In still other embodiments, the INVADER assayreagents further comprise an ARRESTOR molecule (e.g., ARRESTORoligonucleotide).

In some embodiments one or more of the INVADER assay reagents may beprovided in a predispensed format (e.g., premeasured for use in a stepof the procedure without re-measurement or re-dispensing). In someembodiments, selected INVADER assay reagent components are mixed andpredispensed together. In preferred embodiments, predispensed assayreagent components are predispensed and are provided in a reactionvessel (e.g., including, but not limited to, a reaction tube or a well(e.g., a microtiter plate)). In certain preferred embodiments, theINVADER assay reagents are provided in microfluidic devices such asthose described in U.S. Pat. Nos. 6,627,159; 6,720,187; 6,734,401; and6,814,935, as well as U.S. Pat. Pub. 2002/0064885, each of which isherein incorporated by reference in its entirety. In particularlypreferred embodiments, predispensed INVADER assay reagent components aredried down (e.g., desiccated or lyophilized) in a reaction vessel.

In some embodiments, the INVADER assay reagents are provided as a kit.As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.As used herein, the term “fragmented kit” refers to delivery systemscomprising two or more separate containers that each contains asubportion of the total kit components. The containers may be deliveredto the intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains oligonucleotides. The term “fragmented kit” isintended to encompass kits containing Analyte specific reagents (ASR's)regulated under section 520(e) of the Federal Food, Drug, and CosmeticAct, but are not limited thereto. Indeed, any delivery system comprisingtwo or more separate containers that each contains a subportion of thetotal kit components are included in the term “fragmented kit.” Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

In some embodiments, the present invention provides INVADER assayreagent kits comprising one or more of the components necessary forpracticing the present invention. For example, the present inventionprovides kits for storing or delivering the enzymes and/or the reactioncomponents necessary to practice an INVADER assay. The kit may includeany and all components necessary or desired for assays including, butnot limited to, the reagents themselves, buffers, control reagents(e.g., tissue samples, positive and negative control targetoligonucleotides, etc.), solid supports, labels, written and/orpictorial instructions and product information, inhibitors, labelingand/or detection reagents, package environmental controls (e.g., ice,desiccants, etc.), and the like. In some embodiments, the kits provide asub-set of the required components, wherein it is expected that the userwill supply the remaining components. In some embodiments, the kitscomprise two or more separate containers wherein each container houses asubset of the components to be delivered. For example, a first container(e.g., box) may contain an enzyme (e.g., structure specific cleavageenzyme in a suitable storage buffer and container), while a second boxmay contain oligonucleotides (e.g., INVADER oligonucleotides, probeoligonucleotides, control target oligonucleotides, etc.).

In some preferred embodiments, the INVADER assay reagents furthercomprise reagents for detecting a nucleic acid cleavage product. In someembodiments, one or more oligonucleotides in the INVADER assay reagentscomprise a label. In some preferred embodiments, said firstoligonucleotide comprises a label. In other preferred embodiments, saidthird oligonucleotide comprises a label. In particularly preferredembodiments, the reagents comprise a first and/or a thirdoligonucleotide labeled with moieties that produce a fluorescenceresonance energy transfer (FRET) effect.

As used herein, the term “label” refers to any moiety (e.g., chemicalspecies) that can be detected or can lead to a detectable response. Insome preferred embodiments, detection of a label provides quantifiableinformation. Labels can be any known detectable moiety, such as, forexample, a radioactive label (e.g., radionuclides), a ligand (e.g.,biotin or avidin), a chromophore (e.g., a dye or particle that imparts adetectable color), a hapten (e.g., digoxygenin), a mass label, latexbeads, metal particles, a paramagnetic label, a luminescent compound(e.g., bioluminescent, phosphorescent or chemiluminescent labels) or afluorescent compound.

A label may be joined, directly or indirectly, to an oligonucleotide orother biological molecule. Direct labeling can occur through bonds orinteractions that link the label to the oligonucleotide, includingcovalent bonds or non-covalent interactions such as hydrogen bonding,hydrophobic and ionic interactions, or through formation of chelates orcoordination complexes. Indirect labeling can occur through use of abridging moiety or “linker”, such as an antibody or additionaloligonucleotide(s), which is/are either directly or indirectly labeled.

Labels can be used alone or in combination with moieties that cansuppress (e.g., quench), excite, or transfer (e.g., shift) emissionspectra (e.g., fluorescence resonance energy transfer (FRET)) of a label(e.g., a luminescent label).

As used herein, the term “FRET” refers to fluorescence resonance energytransfer, a process in which moeities (e.g., fluorphores) transferenergy (e.g., among themselves, or, from a fluorophore to anon-fluorophore (e.g., a quencher molecule)). In some circumstances,FRET involves an excited donor fluorophore transferring energy to alower-energy acceptor fluorophore via a short-range (e.g., about 10 nmor less) dipole-dipole interaction. In other circumstances, FRETinvolves a loss of fluorescence energy from a donor and an increase influorescence in an acceptor fluorophore. In still other forms of FRET,energy can be exchanged from an excited donor fluorophore to anon-fluorescing molecule (e.g., a quenching molecule). FRET is known tothose of skill in the art and has been described (See, e.g., Stryer etal., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol.,246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res573, 103-110, each of which is incorporated herein by reference in itsentirety).

As used herein, the term “donor” refers to a moiety (e.g., afluorophore) that absorbs at a first wavelength and emits at a second,longer wavelength. The term “acceptor” refers to a moiety such as afluorophore, chromophore, or quencher and that is able to absorb some ormost of the emitted energy from the donor when it is near the donorgroup (typically between 1-100 nm). An acceptor may have an absorptionspectrum that overlaps the donor's emission spectrum. Generally, if theacceptor is a fluorophore, it then re-emits at a third, still longerwavelength; if it is a chromophore or quencher, it releases the energyabsorbed from the donor without emitting a photon. In some preferredembodiments, alteration in energy levels of donor and/or acceptormoieties are detected (e.g., via measuring energy transfer (e.g., bydetecting light emission) between or from donors and/or acceptormoieties). In some preferred embodiments, the emission spectrum of anacceptor moeity is distinct from the emission spectrum of a donor moietysuch that emissions (e.g., of light and/or energy) from the moieties canbe distinguished (e.g., spectrally resolved) from each other.

In some embodiments, a donor moiety is used in combination with multipleacceptor moieties. In a preferred embodiment, a donor moiety is used incombination with a non-fluorescing quencher moiety and with an acceptormoiety, such that when the donor moiety is close (e.g. between 1-100 nm,or more preferably, between 1-25 nm, or even more preferably around 10nm or less) to the quencher, its excitation is transferred to thequencher moiety rather than the acceptor moiety, and when the quenchermoiety is removed (e.g., by cleavage of a probe), donor moietyexcitation is transferred to an acceptor moiety. In some preferredembodiments, emission from the acceptor moiety is detected (e.g., usingwavelength shifting molecular beacons) (See, e.g., Tyagi, et al., NatureBiotechnology 18:1191 (2000); Mhlanga and Malmberg, 2001 Methods 25,463-471; Olivier, 2005 Mutant Res 573, 103-110, and U.S. Pat. App.20030228703, each of which is incorporated herein by reference in itsentirety).

Detection of labels or a detectable response (e.g., provided by thelabels) can be measured using a multitude of techniques, systems andmethods known in the art. For example, a label may be detected becausethe label provides detectable fluorescence (e.g., simple fluorescence,FRET, time-resolved fluorescence, fluorescence quenching, fluorescencepolarization, etc.), radioactivity, chemiluminescence,electrochemiluminescence, RAMAN, colorimetry, gravimetry, hyrbridization(e.g., to a sequence in a hybridization protection assay), X-raydiffraction or absorption, magnetism, enzymatic activity,characteristics of mass or behavior affected by mass (e.g., MALDItime-of-flight mass spectrometry), and the like.

A label may be a charged moiety (positive or negative charge) oralternatively, may be charge neutral. Labels can include or consist ofnucleic acid or protein sequence, so long as the sequence comprising thelabel is detectable. In some embodiments, the label is not nucleic acidor protein.

In some embodiments, a label comprises a particle for detection. Forexample, in some embodiments, the particle is a phosphor particle. Anexample of a phosphor particle includes, but is not limited to, anup-converting phosphor particle (See, e.g., Ostermayer, Preparation andproperties of infrared-to-visible conversion phosphors. Metall. Trans.752, 747-755 (1971)). In some embodiments, rare earth-doped ceramicparticles are used as phosphor particles. Phosphor particles may bedetected by any suitable method, including but not limited toup-converting phosphor technology (UPT), in which up-convertingphosphors transfer low energy infrared (IR) radiation to high-energyvisible light. Although an understanding of the mechanism is notnecessary to practice the present invention and the present invention isnot limited to any particular mechanism of action, in some embodimentsthe UPT up-converts infrared light to visible light by multi-photonabsorption and subsequent emission of dopant-dependant phosphorescence(See, e.g., U.S. Pat. No. 6,399,397; van De Rijke, et al., NatureBiotechnol. 19(3):273-6 (2001); Corstjens, et al., IEEE Proc.Nanobiotechnol. 152(2):64 (2005), each incorporated by reference hereinin its entirety.

As used herein, the term “distinct” in reference to signals (e.g., ofone or more labels) refers to signals that can be differentiated onefrom another, e.g., by spectral properties such as fluorescence emissionwavelength, color, absorbance, mass, size, fluorescence polarizationproperties, charge, etc., or by capability of interaction with anothermoiety, such as with a chemical reagent, an enzyme, an antibody, etc.

It will be apparent to one of skill in the art that there are a largenumber of methods (e.g., analytical procedures) that may be used todetect the presence or absence of a nucleic acid sequence (e.g., a gene(e.g., wild-type, mutant (e.g., comprising one or more variantnucleotides at one or more positions), heterologous, etc.)). Suchmethods include, but are not limited to, nucleic acid discriminationtechniques, amplification reactions and/or a signal generating systems.Such methods include, but are not limited to, DNA sequencing,hybridization sequencing, protein truncation test, single-strandconformation polymorphism analysis (SSCP), denaturing gradient gelelectrophoresis, temperature gradient gel electrophoresis, heteroduplexanalysis, chemical mismatch cleavage, restriction enzyme digestion, andenzymatic mismatch cleavage, solid phase hybridization, dot blots,multiple allele specific diagnostic assays, reverse dot blots,oligonucleotide arrays (e.g., DNA chips), solution phase hybridization(e.g., TAQMAN (See, e.g., U.S. Pat. Nos. 5,210,015 and 5,487,972, eachof which is herein incorporated by reference in its entirety) andmolecular beacons (See, e.g., Tyagi et al. 1996 Nature Biotech, 14, 303and Int. App. WO 95/13399, herein incorporated by reference), extensionbased amplification (e.g., amplification refractory mutation systems,amplification refractory mutation system linear extensions (See, e.g.,EP 332435, herein incorporated by reference in its entirety),competitive oligonucleotide priming system (See, e.g., Gibbs et al.,1989 Nucleic Acids Research 17, 2347, herein incorporated by referencein its entirety), mini sequencing, restriction fragment lengthpolymorphism, restriction site generating PCR, oligonucleotide ligationassay and many others described herein and elsewhere.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of Example 1, where two invasive cleavageassays were used to detect different parts of the same viral sequence.

FIG. 2 shows the results of Example 2, where the primary probe wasprovided at 1× and 0.01×, allowing the assay to detect viral RNA in alinear dynamic range from 50 to 8,000,000 copies.

FIG. 3 shows the results of Example 3, which describe the simultaneousdetection of a first virus-derived target RNA and second virus-derivedtarget DNA with distinguishing fluorescent signals directed to the FAMand Red fluorescent channels, respectively.

FIG. 4 shows the results of Example 4, which describe the simultaneousdetection of a first virus-derived target RNA and second virus-derivedtarget DNA with both assays reporting to the same fluorescent dye.

FIG. 5 shows an overview of one exemplary embodiment of the INVADERassay.

FIG. 6 shows one exemplary embodiment of a T-structure invasive cleavageassay of the present invention. FIG. 6A shows the stem oligonucleotide(SEQ ID NO:6), with its 3′ target specific region hybridized to thehypothetical target sequence (SEQ ID NO:7) and its stem region nothybridized to the target sequence. FIG. 6A also shows the upstreamoligonucleotide (SEQ ID NO:8), with its 5′ target specific regionhybridized to the target sequence and its stem specific regionhybridized to a portion of the stem region of the stem oligonucleotide.FIG. 6A also show the 3′ region of the upstream oligonucleotide as asingle base N in this embodiment. A downstream probe is also shown inFIG. 6A with a 3′ region hybridized to a portion of the stem region ofthe stem oligonucleotide and a 5′ region not hybridized to the stemregion of the stem oligonucleotide. As seen in FIG. 6A, the downstreamprobe, upstream oligonucleotide and stem region of the stemoligonucleotide combine to form an invasive cleavage structure. FIG. 6Ashows a small box around the highlights the area of overlap between thedownstream probe and upstream oligonucleotide on the stem region. FIG.6B shows the result of a cleavage agent recognizing the structure shownin FIG. 6A and cleaving the invasive cleavage structure. As can be seen,the downstream probe is cleaved resulting in a 5′ cleaved portion (SEQID NO:10) and a remainder portion (SEQ ID NO:11). In certainembodiments, the 5′ cleaved portion can be configured to serve as anupstream (INVADER) oligonucleotide with a FRET cassette (e.g. as shownin FIG. 5) in order to generate a detectable signal.

FIG. 7 shows one exemplary embodiment of a T-structure andpolymerization methods that can be used to generate target dependentnon-target amplification. FIG. 7A shows a stem oligonucleotide (SEQ IDNO:12) and an extended upstream oligonucleotide (SEQ ID NO:13)hybridized to a hypothetical target sequence (SEQ ID NO:7). FIG. 7Ashows dashed lines to indicate how the 3′ end of an upstreamoligonucleotide was extended by a polymerase using the stem region ofthe stem oligonucleotide as a template to generate the extended upstreamoligonucleotide. FIG. 7B shows, after that after the extended upstreamoligonucleotide is separated from the target sequence and stemoligonucleotide, it can be hybridized to a primer. In FIG. 7B, theprimer is hybridized to the upstream oligo extended region (the regiongenerated in FIG. 7A as shown in dashed lines). This primer can primepolymerization using the extended upstream oligonucleotide to generate astem amplicon sequence as shown in FIG. 7B (shown as the combination ofthe primer and the extended bases shown in dashed lines). The stemamplicon sequence and extended upstream oligonucleotide can then beseparated (e.g., by heating). As shown in FIG. 7C, the stem amplicon canthen be detected by an invasive cleavage reaction. The stem ampliconsequence and extended upstream oligonucleotides could also be used astemplates for one or more rounds of PCR (not shown in this figure).

FIG. 8 shows the results of a 120 microfluidic card dispensing run usingNanoScreen pipette tips (FIG. 8A) or Beckman pipette tips (FIG. 8B),without also using non-ionic detergent in the nucleic acid mixture.

FIG. 9 shows the result of a 120 microfluidic card dispensing run withBeckman pipette tips using either a water diluent (FIG. 9A) or tRNA(FIG. 9B) in the nucleic acid mixture, without also using a non-ionicdetergent.

FIG. 10 shows the result of a 120 microfluidic card dispensing run withBeckman tips using either a water diluent (FIG. 10A) or water plus 0.25%TWEEN 20 and 0.25% Nonident P40 (FIG. 10B).

FIG. 11 shows the result of a 72 microfluidic card dispensing run withBeckman pipette tips using either a standard water diluent solution(FIG. 11B), water diluent plus 0.25% TWEEN (FIG. 11B), water diluentplus 0.005% TWEEN (FIG. 11C), and water diluent plus 0.0025% TWEEN (FIG.11D).

DESCRIPTION OF THE INVENTION

The present invention provides systems, methods and kits for low-leveldetection of nucleic acids, detecting at least two different viralsequences in a single reaction vessel, and increasing the dynamic rangeof detection of a viral target nucleic acid in a sample. The presentinvention also provides T-structure invasive cleavage assays, as well asT-structure related target dependent non-target amplification methodsand compositions. The present invention further relates to methods,compositions, devices and systems for consistent nucleic acid dispensingonto surfaces.

I. Increased Dynamic Range Detection of Viral Target Sequences

In some embodiments, the present invention achieves greater dynamicrange of detection through the use of differential levels ofamplification of regions of a target nucleic acid, such as a viraltarget nucleic acid (e.g., no amplification, linear amplification at oneor more efficiencies, and/or exponential amplification at one or moreefficiencies). In some embodiments, the present invention achievesgreater dynamic range of detection through the use of probes withdifferent hybridization properties to one or more analyte-specificregions of a target nucleic acid or target nucleic acids (e.g., viraltarget nucleic acids). In some embodiments, the present inventionachieves greater dynamic range of detection through the use of differentsignal generation methods. In some embodiments, the present inventionachieves greater dynamic range of detection through the use of differentsignal detection methods. In preferred embodiments, combinations of twoor more of the methods are employed. For example, in some preferredembodiments, two or more probes (e.g., three, four, etc.) are contactedwith first and second amplicons obtained via different levels ofamplification. In some such embodiments, each probe generates the sametype of signal so that one simply detects total signal generated by thereactions. The collective signal permits detection of target nucleicacid over a broad dynamic range. For example, experiments conductedduring the development of the present invention have demonstrated theability to detect target nucleic acid from samples differing in overeight logs of copy number of target nucleic acid originally present inthe sample.

In certain embodiments, the present invention provides methodologies forexpansion of the dynamic range of hybridization assays, such as serialinvasive cleavage assays. In some embodiments, the upper limit ofdynamic range may be expanded by the use of an additional probe that ispresent in the reaction at a lower concentration than another probe. Insome embodiments, this additional probe will hybridize to the sameregion of the target. For invasive cleavage reactions, this probe maycontain a different arm, or flap, sequence that is released aftercleavage. In certain embodiments related to invasive cleavage assays, asecond FRET cassette will also be added to the reaction with theappropriate sequence to detect those cleaved flaps from the additionalprobe. Generally the concentration of the second FRET cassette is aboutthe same as the first FRET cassette. For example, in certainembodiments, if probe B is present in the reaction at 100-fold lowerconcentration than probe A, this will enable the detection of targetnucleic acid when it is present at concentrations above the upper limitof detection of probe A. In this manner, each additional probe, presentat 100-fold lower concentrations will enable the detection of twoadditional orders of magnitude of probe concentration. This methodologyis not limited to two primary probes, but may be expanded to three ormore. Preferably, the methods are combined with amplification methodswhere one part of the target is amplified to a different level that asecond part of the target.

As mentioned above, in certain embodiments, two probes are employed thatare present at different concentrations that detect the same targetnucleic acid molecule across a broad range of concentrations. In someembodiments involving invasive cleavage reactions, each of the twoprimary probes contain the same analyte specific region (ASR) but havedifferent flap regions. Each of these two flap regions, when cleaved,reports to a different FRET cassette or other reporter sequence orsystem. In some embodiments, the two FRET cassettes both contain thesame fluorophore molecule. In this system, an increase in dynamic rangeis achieved without the use of multiple different fluorophores. Thissystem, therefore, offers a cost advantage over multiple fluorophoresystems. Furthermore, expansion of dynamic range with a singlefluorophore allows for multiplexing with multiple fluorophores fordetection of different targets in the same vessel across a broaderdynamic range than was previously feasible.

In certain embodiments, the concentration of each primary probe ispresent at 100-fold difference relative to each other, and theconcentration of the two FRET cassettes are present at equivalentconcentrations. In certain embodiments, as an example, the dynamic rangewith each of the primary probes present individually may be 10̂4-10̂6 and10̂6-10̂8, respectively, while the dynamic range of the assay when bothare present at the requisite different concentrations may be 10̂4-10̂8.The dynamic range of the serial invasive cleavage assay may be furtherexpanded by the use of further additional primary probes, each presentat different concentrations. In this manner, three, four, five or moreprimary probes, each having the same ASR and different flaps may reportto the same number of different FRET cassettes, each reporting the samecolor or detection format. Such a combination of primary probes enablesthe expansion of the dynamic range to cover 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 orders of magnitude.

The methods of the present invention are not limited by the type oftarget nucleic acid. For example, the target nucleic acid may include,for example, nucleic acid material prepared from viruses having an RNAgenome. Typically, the RNA target sequence will be converted to a cDNAmolecule through the action of a reverse transcriptase, and thendetected by the nucleic acid detection assay. Incorporation of themethods of the present invention will increase the dynamic range ofdetection of RNA target sequences to a breadth not previously feasible.

The methods of the present invention may be combined with amplificationmethods (e.g., PCR) to extend the lower limit of detection down to thetheoretical limit of amplification, on the order of 1 copy per reactionvessel. Using this approach, the dynamic range of nucleic acid detectionassay may be, for example, from 1 to 10̂7 copies using a single set ofreaction conditions and probe combinations in each reaction vessel beingcompared.

Additionally, methods of the present invention involve differentialpre-amplification of target species prior to the detection assay. Incertain embodiments, the use of differential semi-nested PCR usingprimers of different melting temperatures will result in a mixedpopulation of different species, each containing the target regiondetected by the detection assay. The species present in higher numbersin the sample after this step can be detected by the probes present atlower concentration within its dynamic range, and the species present inlower numbers in the sample can be detected by the primary probe presentat higher concentration within its dynamic range, as explained above. Inaddition, a population of target molecules present at differentconcentrations can also be generated by simultaneously combining linearand exponential amplification (or other types of amplification that leadto different levels of amplification). For example, two target-derivedamplicons, both containing the target region detected by the nucleicacid detection assay, would be generated by producing one with a singlePCR primer (for linear amplification) and the other with two PCR primers(for exponential amplification). As above, the different concentrationsof targets can be detected with multiple primary probes tailored todetect those concentrations within their dynamic range. Further,non-amplified and amplified DNA can also be simultaneously detectedusing the above-described combination of probes.

Differential pre-amplification may also comprise multiple similaramplifications (e.g., exponential amplifications) that are performed atdifferent efficiencies so as to allow expanded dynamic range. By way ofexample and not to be limited to any particular embodiment or mechanism,target sequences may be selected such that one region to be amplifiedcomprises few of a selected nucleotide, while another region to beamplified comprises an abundance of the same nucleotide.Pre-amplification under conditions wherein the dNTP required toreplicated the selected nucleotide is limited or omitted will favoramplification of the sequence that is largely free of the limitingnucleotide. Conditions can be selected to allow the other sequence toamplify inefficiently, e.g., by mis-incorporating bases. This is but oneway in which differential pre-amplification can be configured to allow.

In some embodiments, additional probes are used to further expand thedynamic range (e.g., three probes of different concentrations that eachbind to the same analyte-specific region). In some embodiments, themethod detects one or more probes under each of three distinctamplification conditions: e.g., one probe or probe set that detectsexponentially amplified target nucleic acid; one probe or probe set thatdetects linearly amplified target nucleic acid; and one probe or probeset that detects unamplified target nucleic acid. Additionalamplification conditions may also be used (e.g., exponentialamplification using primers or other reaction conditions that providedifferent amplification efficiency per cycle—e.g., a first set that is90% efficient per cycle and a second set that is 70% efficient percycle).

Where PCR or other amplification techniques are used, it may bedesirable to use buffers and other agents and reaction conditions thatminimize limitations of the respective amplification techniques. Forexample, where PCR is used, in some embodiments, a short amplicon isused. In some embodiments, the amplicon is less than one kilobase inlength, although the present invention is not limited to such amplicons.In some embodiments, where the target nucleic is RNA, the amplicon isless than 100 bases, although the present invention is not so limited.

Accordingly, in some embodiments, the present invention provides methodsand compositions for performing probe hybridization assays. In someembodiments, the method utilizes a primary or first probe and preferablyat least one additional probe having different hybridizationcharacteristics with respect to a target sequence than the primaryprobe. In some embodiments, a single probe that provides enhanceddynamic range is utilized. In preferred embodiments, the compositionsand methods of the present invention utilize a combination of two ormore probe oligonucleotides to increase the dynamic range of detectionof the amount of a target nucleic acid present in a sample. In preferredembodiments, combinations of two or more probe oligonucleotides includea mixture of probe oligonucleotides with varying degrees ofhybridization to a target nucleic acid (e.g., frequency of occupation ofa hybridization site). Exemplary probe oligonucleotides of the presentinvention are described in greater detail below.

In some embodiments, three or more probes are used (e.g., four, five,six, etc.). Two or more of the probes may be configured to hybridize tothe same region of the target nucleic acid. However, one or more of theprobes may be configured to hybridize to a second region of the targetnucleic acid or to a different target nucleic acid. In some embodiments,the pluralities of different probes are configured to generate adetectable signal directly or indirectly. In some embodiments, thedifferent probes use the same type of label so that the detected signalis an additive accumulation of the signal from the first and secondprobes. In some such embodiments, the user of the method observes thesignal throughout the broader dynamic range without knowing or needingto know the contribution provided by each type or probe.

Using such systems and methods, detection of a target nucleic acid canbe achieved through a very extensive dynamic range. In some embodiments,this permits detection of target nucleic acids without the need toamplify the target nucleic acid or without the need to extensivelyamplify the target nucleic acid. However, the systems and methods mayfurther be employed with amplification methods, where desired. Asdescribed herein, the systems and methods of the present invention havebeen exemplified with a combination of polymerase chain extensionamplification and invasive cleavage-based detection. Such methodsexperimentally demonstrated successful detection of target nucleic acidshaving over an eight-log difference in starting concentration. Thus, thesystems and methods of the present invention are exceptionally wellsuited to the detection of target nucleic acids whose concentrationdiffers dramatically from sample to sample. For example, patientsinfected with viruses such as HCV and HIV differ greatly the copy numberof virus target nucleic acid present in sample (e.g., blood) from verylow copy (as few as one copy) to very high copy (millions to billions ofcopies or more). The ability of a single detection system tosimultaneously detect viral target nucleic acid throughout this range isgreatly desired. The present invention provides systems and methods thatfind use for such detection.

The compositions and methods are useful for the detection andquantitation of a wide variety of nucleic acid targets. The compositionsand methods of the present invention are particularly useful for thequantitation of viral target nucleic acids (e.g., viral pathogens).Exemplary viral nucleic acids for which a clinical or research need forthe detection of a large range of viral concentrations (e.g., viralload) include, but are not limited to, human immunodeficiency virus(HIV) and other retroviruses, hepatitis C virus (HCV), hepatitis B virus(HBV), hepatitis A virus (HAV), human cytomegalovirus, (CMV), Epsteinbar virus (EBV), human papilloma virus (HPV), herpes simplex virus(HSV), Varicella Zoster Virus (VZV), bacteriophages (e.g., phagelambda), adenoviruses, and lentiviruses. In other embodiments, thecompositions and methods of the present invention find use in thedetection of bacteria (e.g., pathogens or bacteria important incommercial and research applications). Examples include, but are notlimited to, Chlamydia sp., N. gonorrhea, and group B streptococcus.

In some embodiments, the target sequence is a synthetic sequence. Forexample, a fragment generated in an enzymatic reaction (e.g., arestriction fragment, a cleaved flap from an invasive cleavage reaction,etc.) can be considered a target sequence. In some such embodiments, thedetection of such a molecule indirectly detects a separate targetnucleic acid from which the synthetic sequence was generated. Forexample, in an invasive cleavage reaction, a cleaved flap from a primaryreaction may be detected with first and second probes that are FRETcassettes. The FRET cassettes differ in some characteristic (e.g.,length, etc.) such that the cleaved flap differentially hybridizes tothe first and second probes. By using both FRET cassettes (or a third,fourth, etc.), the dynamic range of the reaction is improved.

The quantitation of target nucleic acids using the methods andcompositions of the present invention are utilized in a variety ofclinical and research applications. For example, in some embodiments,the detection assays with increased dynamic range of the presentinvention are utilized in the detection and quantitation of viralpathogens in human samples. The detection assays of the presentinvention are suitable for use with a variety of purified and unpurifiedsamples including, but not limited to, urine, stool, lymph, whole blood,and serum. In preferred embodiments, the detection assays of the presentinvention are suitable for use in the presence of host cells.

In other embodiments, the detection assays of the present invention finduse in research applications including, but not limited to, drugscreening (e.g., for drugs against viral pathogens), animal models ofdisease, and in vitro quantitation of target nucleic acid (e.g.,bacterial, viral, or genomic nucleic acids).

The probe oligonucleotides of the present invention find use in avariety of nucleic acid detection assays including, but not limited to,those described below. It should be understood that any nucleic aciddetection method that employs hybridization can benefit from the systemsand methods of the present invention.

A. Probe Oligonucleotides

In some embodiments, the present invention provides methods for altering(e.g., increasing) the dynamic range of a nucleic acid detection assayby altering probe oligonucleotides. In some embodiments, the presentinvention provides combinations of two or more probe oligonucleotidesfor use in the same detection assay. The present invention is notlimited by the manner in which probes are modified to alterhybridization characteristics. Certain exemplary embodiments areprovided below.

i. Mismatch Probes

In some embodiments, the present invention provides probes with one ormore (preferably one) mismatch with the target sequence. The presentinvention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that the presence of one ormore mismatches allows the probe to bind to the target, but with areduced affinity as compared to a corresponding probe lackingmismatches. This decreases the percent of the time that the mismatchprobe occupies the target site, thus decreasing the signal generated (orincreasing the signal, depending on the detection system used). Thedecrease in signal allows the detection assay to remain linear oraccurate for quantitation at a higher target concentration.

In some embodiments, mismatch probes are utilized in combination withcompletely complementary probes. The completely complementary probesoccupy the target-binding site a higher percentage of the time than themismatch probes and thus generate more signal. The higher signal allowsfor the detection of lower concentrations of target nucleic acid. Theuse of both probes increases the dynamic range of the detection assay.In particular, as described above, it increases the linearity through abroader concentration of target molecules.

Example 1 and FIGS. 1 and 2 demonstrate how the use of mismatch probescan increase the dynamic range of an assay. A combination of match andmismatch probes was used in an INVADER assay to detect target nucleicacids. The mismatch probe increased the dynamic range by up to 16-foldover the use of a single completely complementary probe.

ii. Lower Probe Concentrations

In other embodiments, the present invention provides a combination ofprobe concentrations to increase the dynamic range of a detection assay.In some embodiments a combination of two or more probe oligonucleotides,each of which is at a different concentration, is utilized. The probespresent at a lower concentration generate a lower signal and are thussuitable for detecting higher target concentrations. The probes presentat a higher concentration generate a higher signal and are thus suitablefor detecting a lower concentration of target nucleic acids. Byutilizing two or more probes at a range of concentrations, a broaderdynamic range of target concentrations can be detected.

When probes are attached to a solid surface, lower probe concentrationcan be achieved, in some embodiments, through the use of differentdensities of probes attached to particular detection zones on the solidsurface. For example, a first probe detection zone has a first densityof the probe and a second probe detection zone has a lower density ofthe probe. Detection at the two detection zones provides enhanceddynamic range. In some embodiments, both detection zones generate thesame type of signal and the total signal from the solid surface isdetected (e.g., in real-time) to detect the target nucleic acid throughan expanded dynamic range.

Example 1 and FIGS. 1 and 2 demonstrate how the use of multiple probespresent at different concentrations probes can increase the dynamicrange of an assay. A combination of concentrations of probes was used inan INVADER assay to detect target nucleic acids. The use of multipleconcentrations of probes increased the dynamic range of the assay overthe use of a single probe.

iii. Charge Modified Probes

In other embodiments, the present invention utilizes charge modifiedprobes to alter binding efficiency of probes (See e.g., U.S. Pat. No.6,780,982, herein incorporated by reference in its entirety for allreasons). In some embodiments, the charge modified probes comprise“charge tags.” Positively charged moieties need not always carry apositive charge. As used herein, the term “positively charged moiety”refers to a chemical structure that possesses a net positive chargeunder the reaction conditions of its intended use (e.g., when attachedto a molecule of interest under the pH of the desired reactionconditions). Indeed, in some preferred embodiments of the presentinvention, the positively charged moiety does not carry a positivecharge until it is introduced to the appropriate reaction conditions.This can also be thought of as “pH-dependent” and “pH-independent”positive charges. pH-dependent charges are those that possess the chargeonly under certain pH conditions, while pH-independent charges are thosethat possess a charge regardless of the pH conditions.

The positively charged moieties, or “charge tags,” when attached toanother entity, can be represented by the formula:

X—Y

where X is the entity (e.g., a solid support, a nucleic acid molecule,etc.) and Y is the charge tag. The charge tags can be attached to otherentities through any suitable means (e.g., covalent bonds, ionicinteractions, etc.) either directly or through an intermediate (e.g.,through a linking group). In preferred embodiments, where X is a nucleicacid molecule, the charge tag is attached to either the 3′ or 5′ end ofthe nucleic acid molecule.

The charge tags may contain a variety of components. For example, thecharge tag Y can be represented by the formula:

Y₁—Y₂

where Y₁ comprises a chemical component that provides the positivecharge to the charge tag and where Y₂ is another desired component. Y₂may be, for example, a dye, another chemical component that provides apositive charge to the charge tag, a functional group for attachment ofother molecules to the charge tag, a nucleotide, etc. Where such astructure is attached to another entity, X, either Y₁ or Y₂ may beattached to X.

X—Y₁—Y₂ or X—Y₂—Y₁

The charge tags are not limited to two components. Charge tags maycomprise any number of desired components. For example, the charge tagcan be represented by the formula:

Y₁—Y₂—Y₃—Y_(n) (n=any positive integer).

where any of the Y_(x) groups comprises a chemical component thatprovides the positive charge to the charge tag and where the other Ygroups are any other desired components. For example, in someembodiments, the present invention provides compositions of thestructure:

X—Y₁—Y₂—Y₃—Y₄

where X is an entity attached to the charge tag (e.g., a solid support,a nucleic acid molecule, etc.) and where Y₁ is a dye, Y₂ is a chemicalcomponent that provides the positive charge to the charge, Y₃ is acomponent containing a functional group that allows the attachment ofother molecules, and Y₄ is a second chemical component that provides apositive charge. The identity of each of Y₁-Y₄ can be interchanged(i.e., the present invention is not limited by the order of thecomponents).

The present invention is not limited by the nature of the chemicalcomponents that provides the positive charge to the charge tag. Suchchemical components include, but are not limited to, amines (primary,secondary, and tertiary amines), ammoniums, and phosphoniums. Thechemical components may also comprise chemical complexes that entrap orare otherwise associated with one or more positively charged metal ions.

In preferred embodiments of the present invention, charge tags areattached to nucleic acid molecules (e.g., DNA molecules). The chargetags may be synthesized directly onto a nucleic molecule or may besynthesized, for example, on a solid support or in liquid phase and thenattached to a nucleic acid molecule or any other desired molecule. Insome preferred embodiments of the present invention, charge tags thatare attached to nucleic acid molecules comprise one or more componentssynthesized by H-phosphonate chemistry, by incorporation of novelphosphoramidites, or a combination of both. For example, compositions ofthe present invention include structures such as:

[X]—[Y₁—Y₂—Y₃—Y₄]

where [X] is a nucleic acid molecule and [Y . . . ] is a charge tag. Insome embodiments, Y₁ is a dye, Y₂ is synthesized using H-phosphonatechemistry and comprises a chemical component that provides a positivecharge to the charge tag, Y₃ is a positively charged phosphoramidite,and Y₄ is a nucleotide or polynucleotide. Any of the Y components areinterchangeable with one another.

As discussed above, one or more components of a charge tag can besynthesized using H-phosphonate chemistry. Production of charge tagusing the methods described herein provides a convenient and flexiblemodular approach for the design of a wide variety of charge tags. Sinceits introduction, solid phase H-phosphonate chemistry (B. C. Froehler,Methods in Molecular Biology, 20:33, S. Agrawal, Ed. Humana Press;Totowa, N.J. [1993]) has been recognized as an efficient tool in thechemical synthesis of natural, modified and labeled oligonucleotides andDNA probes. Those skilled in the art know that this approach allows forthe synthesis of the oligonucleotide fragments with a fully modifiedphosphodiester backbone (e.g., oligonucleotide phosphorothioates;Froechler [1993], supra) or the synthesis of oligonucleotide fragmentsin which only specific positions of the phosphodiester backbone aremodified (Agrawal, et al., Proc. Natl. Acad. Sci USA, 85:7079 [1988],Froehler, Tetrahedron Lett. 27:5575 [1986], Froehler, et al., Nucl.Acids Res. 16:4831 [1988]). The use of H-phosphonate chemistry allowsfor the introduction of different types of modifications into theoligonucleotide molecule (Agrawal, et al., Froehler[1986], supra,Letsinger, et al., J. Am. Chem. Soc., 110:4470 [1988], Agrawal andZamecnik, Nucl. Acid Res. 18:5419 [1990], Handong, et al., BioconjugateChem. 8:49 [1997], Vinogradov, et al., Bioconjugate Chem. 7:3 [1995],Schultz, et al., Tetrahedron Lett. 36:8407 [1995]), however thereplacement of the phosphodiester linkage by the phosphoramidate linkageis one of the most frequent changes due to its effectiveness andsynthetic flexibility. Froehler and Letsinger were among first to usethis approach in the synthesis of modified oligonucleotides in whichphosphodiester linkages were fully or partially replaced by thephosphoramidate linkages bearing positively charged groups (e.g.,tertiary amino groups; Froehler [1986], Froehler, et al., [1988], andLetsinger, et al., supra).

In some embodiments of the present invention, charge tags are generatedusing H-phosphonate chemistry. The charge tags may be assembled on theend of a nucleic acid molecule or may be synthesized separately andattached to a nucleic acid molecule. Any suitable phosphorylating agentmay be used in the synthesis of the charge tag. For example, thecomponent to be added may contain the structure:

A-B—P

where A is a protecting group, B is any desired functional group (e.g.,a functional group that provides a positive charge to the charge tag),and P is a chemical group containing phosphorous. In preferredembodiments, B comprises a chemical group that is capable of providing apositive charge to the charge tag. However, in some embodiments B is afunctional group that allows post-synthetic attachment of a positivelycharged group to the charge tag.

In other embodiments, positively charged phosphoramidites (PCP) andneutral phosphoramidites (NP) are utilized to introduce both positivecharge and structure modulation into the synthesized charge-balanced CREprobe (See e.g., U.S. Pat. No. 6,780,982, herein incorporated byreference in its entirety).

Standard coupling protocol with the use of the phosphoramidite reagents(which are compatible with the chemical synthesis of oligonucleotides)is associated with the introduction, into the growing molecule, of onenegative charge per each performed coupling step, due to the formationof the phosphodiester linkage.

iv. Nucleic Acid Modification Agents

The present invention is not limited to the use of charge tags asmodifiers of probe hybridization efficiency. Any internal (e.g., to theprobe) or external agent that alters the hybridization strength of probebinding is suitable for use with the methods and compositions of thepresent invention.

In some embodiments, the present invention provides probes comprisingintercalating agents. Intercalating agents are agents that are capableof inserting themselves between the successive bases in DNA. In someembodiments, intercalating agents alter the binding properties ofnucleic acid probes.

Examples of intercalating agents are known in the art and include, butart limited to, ethidium bromide, psoralen and derivatives, acridines,proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine,daunomycin, chloroquine, distamycin D, chromomycin, homidium,mithramycin, ruthenium polypyridyls, and anthramycin.

In other embodiments, minor groove DNA binding agents are utilized tomodify (e.g., increase or decrease) the hybridization efficiency ofprobes. Examples of minor groove binding agents include, but are notlimited to, duocarmycins (See e.g., Boger, Pure & Appl. Chem., Vol. 66,No. 4, pp. 837-844, herein incorporated by reference in its entirety),netropsin, bisbenzimidazole, aromatic diamidines, lexitropsins,distamycin, and organic dications, based on unfused-aromatic systems(See e.g., U.S. Pat. No. 6,613,787, herein incorporated by reference inits entirety).

In still further embodiments, modified bases are utilized to alter thehybridization efficiency of probes. For example, in some embodiments,modified bases that include charged groups are utilized. Examplesinclude, but are not limited to, the substitution of a “t” nucleotidewith “amino-T” in a probe and other modified nucleotides.

In yet other embodiments, one or more probe nucleotides are modified bythe covalent attachment of groups that alter the hybridizationproperties of the probe. Examples include, but are not limited to, theattachment of amino acids to nucleotides.

In yet other embodiments, probe oligonucleotides with base analogues areutilized to alter the hybridization characteristics of probes. Forexample, in some embodiments, nucleotides that do not form hydrogenbonds but that still participate in base stacking are utilized. Examplesinclude but are not limited to non-polar, aromatic nucleoside analogssuch as 2,4-difluorotoluene and “universal” bases such as 5-nitroindoleand 3-nitropyrrole. In other embodiments, base analogs that retainhydrogen bonding ability are utilized (See e.g., US Patent applicationUS20040106108A1 and WO 04/065550A3, each of which is herein incorporatedby reference in its entirety for all purposes).

v. Probe Length

In yet other embodiments, probe length is altered in order to alter thehybridization characteristics of a probe. For example, in someembodiments, two or more probes that hybridize to the same targetsequence and share the same sequence are utilized. In some embodiments,one of the probes is shorter by one, two, three, or four or more bases.It is preferred that the probes be truncated from one or both ends.Thus, the probes share sequence in all regions except the truncated 3′or 5′ ends. It is contemplated that the shorter probes will anneal withdecreased hybridization efficiency and will thus be useful in thedetection of higher copy numbers of target sequences than the fulllength probe. In preferred embodiments, a combination of full length andtruncated probes is utilized to give the maximum range of targetconcentration detection. In some embodiments, the same length isemployed, but the probe is split into two or more portions connected bylinkers. Such probes hybridize with different affinity depending on avariety of factors, including secondary structure of the target nucleicacid in regions in which the probes or probe fragments hybridize.

vi. Secondary Structure

In some embodiments, probes that comprise secondary structure areutilized to alter the hybridization efficiency of the probe. Forexample, in some embodiments, two or more probes are designed tohybridize the same target sequence. One of the probes is designed tohave minimal secondary structure. Additional probes are designed thatretain target sequence recognition, but that have secondary structure.It is contemplated that the probes with secondary structure will exhibitdecreased hybridization properties and will thus be suitable for thedetection of large copy numbers of target sequence. The combination ofprobes lacking and containing secondary structure serves to detect alarger dynamic range of target nucleic acids than a single probe.Likewise, probes that hybridize to regions of the target nucleic acidthat differ in secondary structure may be used. For example, a probethat has 18 of 18 bases that bind to linear target nucleic acid willhybridize differently than a similar probe shifted two bases over ontarget nucleic acid such that the two bases on the end of the probecorrespond to a region of the target nucleic acid occupied in aninternal hairpin structure or other secondary structure.

vii. Competitor Oligonucleotides

In yet other embodiments, additional oligonucleotides are utilized tomodify hybridization efficiency of probes. For example, in someembodiments, two probes that recognize the same target sequence aredesigned. One of the probes further comprises additional nucleic acidsequence (e.g., at the 3′ or 5′ end) that does not hybridize to thetarget sequence. Competitor oligonucleotides are designed to hybridizeto the extra region. The binding of the competitor oligonucleotidedecreases the hybridization efficiency of the probe to the target. Thecombination of probes with and without competitor binding sequencesserves to detect a larger dynamic range of target nucleic acids than asingle probe.

viii. Reaction Conditions

In still further embodiments, reaction conditions are modified to alterprobe hybridization characteristics. For example, in some embodiments,identical probes are utilized in separate reaction vessels, chamber, orwells. One reaction vessel utilizes “standard” reaction conditions forthe detection assay (e.g., those supplied by the manufacturer or knownin the art). The other reaction vessel comprises altered reactionconditions that increase or decrease the hybridization efficiency of theprobe. Examples of parameters that affect nucleic acid hybridizationconditions include, but are not limited to, ionic strength, buffercomposition, pH, and additives (e.g., glycerol, polyethylene glycol,proteins).

ix. Stacking Oligonucleotides

In still further embodiments, adjacently hybridizing oligonucleotidesare used to alter probe hybridization characteristics. When shortstrands of nucleic acid align contiguously along a longer strand, thehybridization of each is stabilized by the hybridization of theneighboring fragments because the base pairs can stack along the helixas though the backbone was, in fact, uninterrupted. This cooperativityof binding can give each segment a stability of interaction in excess ofwhat would be expected for the segment hybridizing to the longer nucleicacid alone. In the event of a perturbation in the cooperative binding,e.g., by a mismatch at or near the junction between the contiguousduplexes, this cooperativity can be reduced or eliminated. In someembodiments of the present invention, probes are configured to cooperatein distinct ways with one or more adjacently hybridizingoligonucleotides, so as to provide probes having different hybridizationcharacteristics. In some embodiments, a probe comprises one or moremismatched bases at near the junction with the adjacent oligonucleotide,so as to alter or disrupt cooperativity of binding, as compared to aprobe lacking the mismatches. In other embodiments, a probe comprisesone or more base analogs selected to reduce stacking interactions withadjacent bases. In yet other embodiments, it is envisioned that gaps ofone or more nucleotides (e.g., by the use of truncated probes) are usedto alter cooperativity and thus alter hybridization characteristics. Theuse of a combination of probes that have a range of cooperativities ofbinding with an adjacently hybridized oligonucleotide, and thus having arange of different hybridization stabilities on the target, serves todetect a larger dynamic range of target nucleic acids than a singleprobe.

x. Multiplex Assays

In some embodiments utilizing multiple nucleic acid probes, the probesare utilized in a biplex or multiplex assay in which a plurality ofprobes is included in the same reaction vessel. In some embodiments,each probe in a biplex assay comprises a differently detectable label.For example, some embodiments, each probe in a set comprises a differentfluorescent label that fluoresces at a different wavelength. Many knownprobe binding assays are suitable for use in a multiplex format. Methodsfor performing multiplex assays that are unique to the particular assayformat are described below.

xi. Others

Any other method for altering the hybridization of characteristics of aprobe may be used with the present invention. Other examples include,but are not limited to: use of sequences in probes or targets thatrender the sequence susceptible to differential hybridization behaviorin response to buffer conditions (e.g., the use of guanosine-quartets)or protein/nucleic acid interactions (e.g., by creating binding sitesfor nucleic acid binding proteins or enzyme that bind or alter nucleicacid sequences); use of dangling ends (e.g., for dangling-endstabilization and stacking); attachment of iron or other magnetic agentsto allow concentration of the nucleic acid in a magnetic field; use ofagents that titrate out a specific probe; and the like.

One may also use different labeling techniques to achieve a differentialdetection of signal, independent of the hybridization properties of theprobe. For example, the location of labels and quenchers in a FRETdetection system may be altered between first and second probes to alterthe amount of signal detected from the probes. FRET signaling can alsobe affected by many other parameters, including, but not limited to, theuse of additional chemical moieties that influence the amount ofquenching and the use of secondary structure in the probes. Additionalmethods for altering signal detection include the use of a helperoligonucleotide that is provided at low concentration, that when boundto a target occupied by a probe of the invention, changes the wavelengthor otherwise alters the detectable aspects of the probe. Theconcentration of the helper can be configured to only allow detectionthe alteration when a particular threshold level of probe is hybridizedto target. Any method or system that permits differential detection ofhybridization events may be used in the systems and methods of thepresent invention.

B. Detection Assays

The present invention is not limited to a particular detection assay.Any number of suitable detection assays may be utilized. In someembodiments, the present invention provides methods and compositions forthe detection of DNA or RNA (e.g., viral RNA). In some embodiments, thedetection assays described below are suitable for direct detection ofRNA. In other embodiments, RNA is reverse transcribed (e.g., using areverse transcriptase enzyme such as AMV or MMLV) into DNA and thedetection assay is performed on the corresponding DNA. Methods forreverse transcription are known in the art. In some embodiments, asingle enzyme having both reverse transcriptase and polymeraseactivities is used.

Exemplary assays that find use with the methods of the present inventionare described below.

i. Invasive Cleavage Assays

In some embodiments, the methods and compositions of the presentinvention are used to increase the dynamic range of invasive cleavageassays, such as the INVADER assay. The INVADER assay provides means forforming a nucleic acid cleavage structure that is dependent upon thepresence of a target nucleic acid and cleaving the nucleic acid cleavagestructure so as to release distinctive cleavage products. 5′ nucleaseactivity, for example, is used to cleave the target-dependent cleavagestructure and the resulting cleavage products are indicative of thepresence of specific target nucleic acid sequences in the sample. Whentwo strands of nucleic acid, or oligonucleotides, both hybridize to atarget nucleic acid strand such that they form an overlapping invasivecleavage structure, as described below, invasive cleavage can occur.Through the interaction of a cleavage agent (e.g., a 5′ nuclease) andthe upstream oligonucleotide, the cleavage agent can be made to cleavethe downstream oligonucleotide at an internal site in such a way that adistinctive fragment is produced. Such embodiments have been termed theINVADER assay (Third Wave Technologies, Madison, Wis.) and are describedin U.S. Pat. Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, and6,090,543, WO 97/27214, WO 98/42873, Lyamichev et al., Nat. Biotech.,17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), each of which isherein incorporated by reference in their entirety for all purposes.

The INVADER assay detects hybridization of probes to a target byenzymatic cleavage of specific structures by structure specific enzymes(See, INVADER assays, Third Wave Technologies; See e.g., U.S. Pat. Nos.5,846,717; 6,090,543; 6,001,567; 5,985,557; 6,090,543; 5,994,069;Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA,97:8272 (2000), WO97/27214 and WO98/42873, each of which is hereinincorporated by reference in their entirety for all purposes).

The INVADER assay detects specific DNA and RNA sequences by usingstructure-specific enzymes (e.g. FEN endonucleases) to cleave a complexformed by the hybridization of overlapping oligonucleotide probes.Elevated temperature and an excess of one of the probes enable multipleprobes to be cleaved for each target sequence present withouttemperature cycling. In some embodiments, these cleaved probes thendirect cleavage of a second labeled probe. The secondary probeoligonucleotide can be 5′-end labeled with fluorescent that is quenchedby an internal dye. Upon cleavage, the de-quenched fluorescent labeledproduct may be detected using a standard fluorescence plate reader.

The INVADER assay detects specific target sequences in unamplified, aswell as amplified, RNA and DNA including genomic DNA. In the embodimentsshown schematically in FIG. 5, the INVADER assay uses two cascadingsteps (a primary and a secondary reaction) both to generate and then toamplify the target-specific signal. For convenience, the alleles in thefollowing discussion are described as wild-type (WT) and mutant (MT),even though this terminology does not apply to all genetic variations ortarget sequences. In the primary reaction (FIG. 5, panel A), the WTprimary probe and the INVADER oligonucleotide hybridize in tandem to thetarget nucleic acid to form an overlapping structure. An unpaired “flap”is included on the 5′ end of the WT primary probe. A structure-specificenzyme (e.g. the CLEAVASE enzyme, Third Wave Technologies) recognizesthe overlap and cleaves off the unpaired flap, releasing it as atarget-specific product. In the secondary reaction, this cleaved productserves as an INVADER oligonucleotide on the WT fluorescence resonanceenergy transfer (WT-FRET) probe to again create the structure recognizedby the structure specific enzyme (panel A). When the two dyes on asingle FRET probe are separated by cleavage (indicated by the arrow inFIG. 5), a detectable fluorescent signal above background fluorescenceis produced. Consequently, cleavage of this second structure results inan increase in fluorescence, indicating the presence of the WT allele(or mutant allele if the assay is configured for the mutant allele togenerate the detectable signal). In some embodiments, FRET probes havingdifferent labels (e.g. resolvable by difference in emission orexcitation wavelengths, or resolvable by time-resolved fluorescencedetection) are provided for each allele or locus to be detected, suchthat the different alleles or loci can be detected in a single reaction.In such embodiments, the primary probe sets and the different FRETprobes may be combined in a single assay, allowing comparison of thesignals from each allele or locus in the same sample.

If the primary probe oligonucleotide and the target nucleotide sequencedo not match perfectly at the cleavage site (e.g., as with the MTprimary probe and the WT target, FIG. 5, panel B), the overlappedstructure does not form and cleavage is suppressed. The structurespecific enzyme (e.g., CLEAVASE VIII enzyme, Third Wave Technologies)used cleaves the overlapped structure more efficiently (e.g. at least340-fold) than the non-overlapping structure, allowing excellentdiscrimination of the alleles.

The probes turn over without temperature cycling to produce many signalsper target (i.e., linear signal amplification). Similarly, eachtarget-specific product can enable the cleavage of many FRET probes.

The primary INVADER assay reaction is directed against the target DNA orRNA being detected. The target DNA is the limiting component in thefirst invasive cleavage, since the INVADER and primary probe aresupplied in molar excess. In the second invasive cleavage, it is thereleased flap that is limiting. When these two cleavage reactions areperformed sequentially, the fluorescence signal from the compositereaction accumulates linearly with respect to the target DNA amount.

In certain embodiments, the INVADER assay, or other nucleotide detectionassays, are performed with accessible site designed oligonucleotidesand/or bridging oligonucleotides. Such methods, procedures andcompositions are described in U.S. Pat. No. 6,194,149, WO9850403, andWO0198537, all of which are specifically incorporated by reference intheir entireties.

In certain embodiments, the target nucleic acid sequence is amplifiedprior to detection (e.g. such that synthetic nucleic acid is generated).In some embodiments, the target nucleic acid comprises genomic DNA. Inother embodiments, the target nucleic acid comprises synthetic DNA orRNA. In some preferred embodiments, synthetic DNA within a sample iscreated using a purified polymerase. In some preferred embodiments,creation of synthetic DNA using a purified polymerase comprises the useof PCR. In other preferred embodiments, creation of synthetic DNA usinga purified DNA polymerase, suitable for use with the methods of thepresent invention, comprises use of rolling circle amplification, (e.g.,as in U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, hereinincorporated by reference in their entireties). In other preferredembodiments, creation of synthetic DNA comprises copying genomic DNA bypriming from a plurality of sites on a genomic DNA sample. In someembodiments, priming from a plurality of sites on a genomic DNA samplecomprises using short (e.g., fewer than about 8 nucleotides)oligonucleotide primers. In other embodiments, priming from a pluralityof sites on a genomic DNA comprises extension of 3′ ends in nicked,double-stranded genomic DNA (i.e., where a 3′ hydroxyl group has beenmade available for extension by breakage or cleavage of one strand of adouble stranded region of DNA). Some examples of making synthetic DNAusing a purified polymerase on nicked genomic DNAs, suitable for usewith the methods and compositions of the present invention, are providedin U.S. Pat. Nos. 6,117,634, issued Sep. 12, 2000, and 6,197,557, issuedMar. 6, 2001, and in PCT application WO 98/39485, each incorporated byreference herein in their entireties for all purposes.

In some embodiments, synthetic DNA suitable for use with the methods andcompositions of the present invention is made using a purifiedpolymerase on multiply-primed genomic or other DNA, as provided, e.g.,in U.S. Pat. Nos. 6,291,187, and 6,323,009, and in PCT applications WO01/88190 and WO 02/00934, each herein incorporated by reference in theirentireties for all purposes. In these embodiments, amplification of DNAsuch as genomic DNA is accomplished using a DNA polymerase, such as thehighly processive (d 29 polymerase (as described, e.g., in U.S. Pat.Nos. 5,198,543 and 5,001,050, each herein incorporated by reference intheir entireties for all purposes) in combination withexonuclease-resistant random primers, such as hexamers.

The present invention further provides assays in which the targetnucleic acid is reused or recycled during multiple rounds ofhybridization with oligonucleotide probes and cleavage of the probeswithout the need to use temperature cycling (i.e., for periodicdenaturation of target nucleic acid strands) or nucleic acid synthesis(i.e., for the polymerization-based displacement of target or probenucleic acid strands). When a cleavage reaction is run under conditionsin which the probes are continuously replaced on the target strand (e.g.through probe-probe displacement or through an equilibrium betweenprobe/target association and disassociation, or through a combinationcomprising these mechanisms, (Reynaldo et al., J. Mol. Biol. 97: 511-520(2000)), multiple probes can hybridize to the same target, allowingmultiple cleavages, and the generation of multiple cleavage products.

As described above, in some embodiments, the present invention providesmethods of utilizing the INVADER assay to quantitate the amount oftarget nucleic present in a sample. In some embodiments, the dynamicrange of INVADER assays is increased using mismatch probes, alone or incombination with completely homologous probes. It is preferred that themismatch is not present at the site of cleavage by the cleavage enzyme.In other embodiments, dynamic range of the INVADER assay is increased byusing probes of multiple concentrations. In preferred embodiments, eachprobe in a multiple probe INVADER assay comprises a different label,allowing the reactions to be run in the same well or tube of thereaction vessel and detected simultaneously. However, the probes mayalso share the same label, permitting the combined signal to beinterpreted as one detection event. In some preferred embodiments, areal time assay, in which signal is measured continuously or at timeintervals, is utilized. In other embodiments, a single end-pointdetection is taken at a desired time point. In yet other embodiment, twoor more time point readings are taken.

In other embodiments, composite or split probe oligonucleotides areutilized to increase the dynamic range is utilized in theINVADER-directed cleavage assay. For example, the probe oligonucleotidemay be split into two oligonucleotides that anneal in a contiguous andadjacent manner along a target oligonucleotide. The probeoligonucleotide is assembled from two smaller pieces: a short segment of6-10 nts (termed the “miniprobe”), that is to be cleaved in the courseof the detection reaction, and an oligonucleotide that hybridizesimmediately downstream of the miniprobe (termed the “stacker”), thatserves to stabilize the hybridization of the probe. To form the cleavagestructure, an upstream oligonucleotide (the INVADER oligonucleotide) isprovided to direct the cleavage activity to the desired region of theminiprobe. Assembly of the probe from non-linked pieces of nucleic acid(i.e., the miniprobe and the stacker) allows regions of sequences to bechanged without requiring the re-synthesis of the entire provensequence, thus improving the cost and flexibility of the detectionsystem. In addition, the use of unlinked composite oligonucleotidesmakes the system more stringent in its requirement of perfectly matchedhybridization to achieve signal generation, allowing this to be used asa sensitive means of detecting mutations or changes in the targetnucleic acid sequences. In some embodiments, two probe/stacker designsare utilized to increase the dynamic range of the assay. A firstconfiguration, without a gap between the probe and the stacker isutilized. This configuration occupies the target site at a highfrequency and serves to generate a higher signal (e.g., in the presenceof a low concentration of target). A second configuration, in which asingle nucleotide gap between the probe and stacker oligonucleotide isintroduced, it utilized for the detection of high concentrations oftarget. The gapped configuration probe and stacker oligonucleotideshybridize at a lower strength and thus occupy the target site at a lowerfrequency. This generates a lower signal, which is useful in thedetection of high amounts of target sequences.

Additional considerations for performing the INVADER assay are discussedin more detail below.

Oligonucleotide Design for the INVADER Assay

In some embodiments where an oligonucleotide is designed for use in theINVADER assay to detect a target nucleic acid, the sequence(s) ofinterest are entered into the INVADERCREATOR program (Third WaveTechnologies, Madison, Wis.). Sequences may be input for analysis fromany number of sources, either directly into the computer hosting theINVADERCREATOR program, or via a remote computer linked through acommunication network (e.g., a LAN, Intranet or Internet network). Theprogram designs probes for both the sense and antisense strand. Strandselection is generally based upon the ease of synthesis, minimization ofsecondary structure formation, and manufacturability. In someembodiments, the user chooses the strand for sequences to be designedfor. In other embodiments, the software automatically selects thestrand. By incorporating thermodynamic parameters for optimum probecycling and signal generation (Allawi and SantaLucia, Biochemistry,36:10581 [1997]), oligonucleotide probes may be designed to operate at apre-selected assay temperature (e.g., 63° C.). Based on these criteria,a final probe set (e.g., match and mismatch probes and an INVADERoligonucleotide) is selected.

In some embodiments, the INVADERCREATOR system is a web-based programwith secure site access that contains a link to BLAST (available at theNational Center for Biotechnology Information, National Library ofMedicine, National Institutes of Health website) and that can be linkedto RNAstructure (Mathews et al., RNA 5:1458 [1999]), a software programthat incorporates mfold (Zuker, Science, 244:48 [1989]). RNAstructuretests the proposed oligonucleotide designs generated by INVADERCREATORfor potential uni- and bimolecular complex formation. INVADERCREATOR isopen database connectivity (ODBC)-compliant and uses the Oracle databasefor export/integration. The INVADERCREATOR system was configured withOracle to work well with UNIX systems, as most genome centers areUNIX-based.

In some embodiments, the INVADERCREATOR analysis is provided on aseparate server (e.g., a Sun server) so it can handle analysis of largebatch jobs. For example, a customer can submit up to 2,000 SNP sequencesin one email. The server passes the batch of sequences on to theINVADERCREATOR software, and, when initiated, the program designsdetection assay oligonucleotide sets. In some embodiments, probe setdesigns are returned to the user within 24 hours of receipt of thesequences.

Each INVADER reaction includes at least two target sequence-specific,unlabeled oligonucleotides for the primary reaction: an upstream INVADERoligonucleotide and a downstream Probe oligonucleotide. The INVADERoligonucleotide is generally designed to bind stably at the reactiontemperature, while the probe is designed to freely associate anddisassociate with the target strand, with cleavage occurring only whenan uncut probe hybridizes adjacent to an overlapping INVADERoligonucleotide. In some embodiments, the probe includes a 5′ flap or“arm” that is not complementary to the target, and this flap is releasedfrom the probe when cleavage occurs. In some embodiments, the releasedflap participates as an INVADER oligonucleotide in a secondary reaction.

The following discussion provides one example of how a user interfacefor an INVADERCREATOR program may be configured.

The user opens a work screen, e.g., by clicking on an icon on a desktopdisplay of a computer (e.g., a Windows desktop). The user entersinformation related to the target sequence for which an assay is to bedesigned. In some embodiments, the user enters a target sequence. Inother embodiments, the user enters a code or number that causesretrieval of a sequence from a database. In still other embodiments,additional information may be provided, such as the user's name, anidentifying number associated with a target sequence, and/or an ordernumber. In preferred embodiments, the user indicates (e.g. via a checkbox or drop down menu) that the target nucleic acid is DNA or RNA. Inother preferred embodiments, the user indicates the species from whichthe nucleic acid is derived. In particularly preferred embodiments, theuser indicates whether the design is for monoplex (i.e., one targetsequence or allele per reaction) or multiplex (i.e., multiple targetsequences or alleles per reaction) detection. When the requisite choicesand entries are complete, the user starts the analysis process. In oneembodiment, the user clicks a “Go Design It” button to continue.

In some embodiments, the software validates the field entries beforeproceeding. In some embodiments, the software verifies that any requiredfields are completed with the appropriate type of information. In otherembodiments, the software verifies that the input sequence meetsselected requirements (e.g., minimum or maximum length, DNA or RNAcontent). If entries in any field are not found to be valid, an errormessage or dialog box may appear. In preferred embodiments, the errormessage indicates which field is incomplete and/or incorrect. Once asequence entry is verified, the software proceeds with the assay design.

In some embodiments, the information supplied in the order entry fieldsspecifies what type of design will be created. In preferred embodiments,the target sequence and multiplex check box specify which type of designto create. Design options include but are not limited to SNP assay,Multiplexed SNP assay (e.g., wherein probe sets for different allelesare to be combined in a single reaction), Multiple SNP assay (e.g.,wherein an input sequence has multiple sites of variation for whichprobe sets are to be designed), and Multiple Probe Arm assays.

In some embodiments, the INVADERCREATOR software is started via a WebOrder Entry (WebOE) process (i.e., through an Intra/Internet browserinterface) and these parameters are transferred from the WebOE viaapplet <param> tags, rather than entered through menus or check boxes.

In the case of Multiple SNP Designs, the user chooses two or moredesigns to work with. In some embodiments, this selection opens a newscreen view (e.g., a Multiple SNP Design Selection view). In someembodiments, the software creates designs for each locus in the targetsequence, scoring each, and presents them to the user in this screenview. The user can then choose any two designs to work with. In someembodiments, the user chooses a first and second design (e.g., via amenu or buttons) and clicks a “Go Design It” button to continue.

To select a probe sequence that will perform optimally at a pre-selectedreaction temperature, the melting temperature (T_(m)) of the SNP to bedetected is calculated using the nearest-neighbor model and publishedparameters for DNA duplex formation (Allawi and SantaLucia,Biochemistry, 36:10581 [1997]). In embodiments wherein the target strandis RNA, parameters appropriate for RNA/DNA heteroduplex formation may beused. Because the assay's salt concentrations are often different thanthe solution conditions in which the nearest-neighbor parameters wereobtained (1M NaCl and no divalent metals), and because the presence andconcentration of the enzyme influence optimal reaction temperature, anadjustment should be made to the calculated T_(m) to determine theoptimal temperature at which to perform a reaction. One way ofcompensating for these factors is to vary the value provided for thesalt concentration within the melting temperature calculations. Thisadjustment is termed a ‘salt correction’. As used herein, the term “saltcorrection” refers to a variation made in the value provided for a saltconcentration for the purpose of reflecting the effect on a T_(m)calculation for a nucleic acid duplex of a non-salt parameter orcondition affecting said duplex. Variation of the values provided forthe strand concentrations will also affect the outcome of thesecalculations. By using a value of 0.5 M NaCl (SantaLucia, Proc Natl AcadSci USA, 95:1460 [1998]) and strand concentrations of about 1 mM of theprobe and 1 fM target, the algorithm for used for calculatingprobe-target melting temperature has been adapted for use in predictingoptimal INVADER assay reaction temperature. For a set of 30 probes, theaverage deviation between optimal assay temperatures calculated by thismethod and those experimentally determined is about 1.5° C.

The length of the downstream probe to a given target sequence is definedby the temperature selected for running the reaction (e.g., 63° C.).Starting from the position of the variant nucleotide on the target DNA(the target base that is paired to the probe nucleotide 5′ of theintended cleavage site), and adding on the 3′ end, an iterativeprocedure is used by which the length of the target-binding region ofthe probe is increased by one base pair at a time until a calculatedoptimal reaction temperature (T_(m) plus salt correction to compensatefor enzyme effect) matching the desired reaction temperature is reached.The non-complementary arm of the probe is preferably selected to allowthe secondary reaction to cycle at the same reaction temperature. Theentire probe oligonucleotide is screened using programs such as mfold(Zuker, Science, 244: 48 [1989]) or Oligo 5.0 (Rychlik and Rhoads,Nucleic Acids Res, 17: 8543 [1989]) for the possible formation of dimercomplexes or secondary structures that could interfere with thereaction. The same principles are also followed for INVADERoligonucleotide design. Briefly, starting from the position N on thetarget DNA, the 3′ end of the INVADER oligonucleotide is designed tohave a nucleotide not complementary to either allele suspected of beingcontained in the sample to be tested. The mismatch does not adverselyaffect cleavage (Lyamichev et al., Nature Biotechnology, 17: 292[1999]), and it can enhance probe cycling, presumably by minimizingcoaxial stabilization effects between the two probes. Additionalresidues complementary to the target DNA starting from residue N−1 arethen added in the 5′ direction until the stability of the INVADERoligonucleotide-target hybrid exceeds that of the probe (and thereforethe planned assay reaction temperature), generally by 15-20° C.

It is one aspect of the assay design that the all of the probe sequencesmay be selected to allow the primary and secondary reactions to occur atthe same optimal temperature, so that the reaction steps can runsimultaneously. In an alternative embodiment, the probes may be designedto operate at different optimal temperatures, so that the reaction stepsare not simultaneously at their temperature optima.

In some embodiments, the software provides the user an opportunity tochange various aspects of the design including but not limited to:probe, target and INVADER oligonucleotide temperature optima andconcentrations; blocking groups; probe arms; dyes, capping groups andother adducts; individual bases of the probes and targets (e.g., addingor deleting bases from the end of targets and/or probes, or changinginternal bases in the INVADER and/or probe and/or targetoligonucleotides). In some embodiments, changes are made by selectionfrom a menu. In other embodiments, changes are entered into text ordialog boxes. In preferred embodiments, this option opens a new screen(e.g., a Designer Worksheet view).

In some embodiments, the software provides a scoring system to indicatethe quality (e.g., the likelihood of performance) of the assay designs.In one embodiment, the scoring system includes a starting score ofpoints (e.g., 100 points) wherein the starting score is indicative of anideal design, and wherein design features known or suspected to have anadverse affect on assay performance are assigned penalty values. Penaltyvalues may vary depending on assay parameters other than the sequences,including but not limited to the type of assay for which the design isintended (e.g., monoplex, multiplex) and the temperature at which theassay reaction will be performed. The following example provides anillustrative scoring criteria for use with some embodiments of theINVADER assay based on an intelligence defined by experimentation.Examples of design features that may incur score penalties include butare not limited to the following [penalty values are indicated inbrackets, first number is for lower temperature assays (e.g., 62-64°C.), second is for higher temperature assays (e.g., 65-66° C.)]:

1. [100:100] 3′ end of INVADER oligonucleotide resembles the probe arm:

ARM SEQUENCE: PENALTY AWARDED ENDS IN: IF INVADER Arm 1 (SEQ ID NO: 1): 5′ . . . GAGGX or CGCGCCGAGG 5′ . . . GAGGXX Arm 2 (SEQ ID NO: 2):  5′. . . CAGACX or ATGACGTGGCAGAC 5′ . . . CAGACXX Arm 3 (SEQ ID NO: 3): 5′ . . . GGAGX or ACGGACGCGGAG 5′ . . . GGAGXX Arm 4 (SEQ ID NO: 4):  5′. . . GTCCX or TCCGCGCGTCC 5′ . . . GTCCXX2. [70:70] a probe has 5-base stretch (i.e., 5 of the same base in arow) containing the polymorphism;3. [60:60] a probe has 5-base stretch adjacent to the polymorphism;4. [50:50] a probe has 5-base stretch one base from the polymorphism;5. [40:40] a probe has 5-base stretch two bases from the polymorphism;6. [50:50] probe 5-base stretch is of Gs—additional penalty;7. [100:100] a probe has 6-base stretch anywhere;8. [90:90] a two or three base sequence repeats at least four times;9. [100:100] a degenerate base occurs in a probe;10. [60:90] probe hybridizing region is short (13 bases or less fordesigns 65-67° C.; 12 bases or less for designs 62-64° C.)11. [40:90] probe hybridizing region is long (29 bases or more fordesigns 65-67° C., 28 bases or more for designs 62-64° C.)12. [5:5] probe hybridizing region length—per base additional penalty13. [80:80] Ins/Del design with poor discrimination in first 3 basesafter probe arm14. [100:100] calculated INVADER oligonucleotide Tm within 7.5° C. ofprobe target Tm (designs 65-67° C. with INVADER oligonucleotide lessthan ≦70.5° C., designs 62-64° C. with INVADER oligonucleotide ≦69.5° C.15. [20:20] calculated probes Tms differ by more than 2.0° C.16. [100:100] a probe has calculated Tm 2° C. less than its target Tm17. [10:10] target of one strand 8 bases longer than that of otherstrand18. [30:30] INVADER oligonucleotide has 6-base stretch anywhere—initialpenalty19. [70:70] INVADER oligonucleotide 6-base stretch is of Gs—additionalpenalty20. [15:15] probe hybridizing region is 14, 15 or 24-28 bases long(65-67° C.) or 13,14 or 26,27 bases long (62-64° C.)21. [15:15] a probe has a 4-base stretch of Gs containing thepolymorphism

In particularly preferred embodiments, temperatures for each of theoligonucleotides in the designs are recomputed and scores are recomputedas changes are made. In some embodiments, score descriptions can be seenby clicking a “descriptions” button. In some embodiments, a BLAST searchoption is provided. In preferred embodiments, a BLAST search is done byclicking a “BLAST Design” button. In some embodiments, this actionbrings up a dialog box describing the BLAST process. In preferredembodiments, the BLAST search results are displayed as a highlighteddesign on a Designer Worksheet.

In some embodiments, a user accepts a design by clicking an “Accept”button. In other embodiments, the program approves a design without userintervention. In preferred embodiments, the program sends the approveddesign to a next process step (e.g., into production; into a file ordatabase). In some embodiments, the program provides a screen view(e.g., an Output Page), allowing review of the final designs created andallowing notes to be attached to the design. In preferred embodiments,the user can return to the Designer Worksheet (e.g., by clicking a “GoBack” button) or can save the design (e.g., by clicking a “Save It”button) and continue (e.g., to submit the designed oligonucleotides forproduction).

In some embodiments, the program provides an option to create a screenview of a design optimized for printing (e.g., a text-only view) orother export (e.g., an Output view). In preferred embodiments, theOutput view provides a description of the design particularly suitablefor printing, or for exporting into another application (e.g., bycopying and pasting into another application). In particularly preferredembodiments, the Output view opens in a separate window.

The present invention is not limited to the use of the INVADERCREATORsoftware. Indeed, a variety of software programs are contemplated andare commercially available, including, but not limited to GCG WisconsinPackage (Genetics computer Group, Madison, Wis.) and Vector NTI(Informax, Rockville, Md.). Other detection assays may be used in thepresent invention.

Multiplex Reactions

Since its introduction in 1988 (Chamberlain, et al. Nucleic Acids Res.,16:11141 (1988)), multiplex PCR has become a routine means of amplifyingmultiple genetic loci in a single reaction. This approach has foundutility in a number of research, as well as clinical, applications.Multiplex PCR has been described for use in diagnostic virology(Elnifro, et al. Clinical Microbiology Reviews, 13: 559 (2000)),paternity testing (Hidding and Schmitt, Forensic Sci. Int., 113: 47(2000); Bauer et al., Int. J. Legal Med. 116: 39 (2002)),preimplantation genetic diagnosis (Ouhibi, et al., Curr Womens HealthRep. 1: 138 (2001)), microbial analysis in environmental and foodsamples (Rudi et al., Int J Food Microbiology, 78: 171 (2002)), andveterinary medicine (Zarlenga and Higgins, Vet Parasitol. 101: 215(2001)), among others. Most recently, expansion of genetic analysis towhole genome levels, particularly for single nucleotide polymorphisms,or SNPs, has created a need for highly multiplexed PCR capabilities.Comparative genome-wide association and candidate gene studies requirethe ability to genotype between 100,000-500,000 SNPs per individual(Kwok, Molecular Medicine Today, 5: 538-5435 (1999); Kwok,Pharmacogenomics, 1: 231 (2000); Risch and Merikangas, Science, 273:1516 (1996)). Moreover, SNPs in coding or regulatory regions alter genefunction in important ways (Cargill et al. Nature Genetics, 22: 231(1999); Halushka et al., Nature Genetics, 22: 239 (1999)), making theseSNPs useful diagnostic tools in personalized medicine (Hagmann, Science,285: 21 (1999); Cargill et al. Nature Genetics, 22: 231 (1999); Halushkaet al., Nature Genetics, 22: 239 (1999)). Likewise, validating themedical association of a set of SNPs previously identified for theirpotential clinical relevance as part of a diagnostic panel will meantesting thousands of individuals for thousands of markers at a time.

Despite its broad appeal and utility, several factors complicatemultiplex PCR amplification. Chief among these is the phenomenon of PCRor amplification bias, in which certain loci are amplified to a greaterextent than others. Two classes of amplification bias have beendescribed. One, referred to as PCR drift, is ascribed to stochasticvariation in such steps as primer annealing during the early stages ofthe reaction (Polz and Cavanaugh, Applied and EnvironmentalMicrobiology, 64: 3724 (1998)), is not reproducible, and may be moreprevalent when very small amounts of target molecules are beingamplified (Walsh et al., PCR Methods and Applications, 1: 241 (1992)).The other, referred to as PCR selection, pertains to the preferentialamplification of some loci based on primer characteristics, ampliconlength, G-C content, and other properties of the genome (Polz, supra).

Another factor affecting the extent to which PCR reactions can bemultiplexed is the inherent tendency of PCR reactions to reach a plateauphase. The plateau phase is seen in later PCR cycles and reflects theobservation that amplicon generation moves from exponential topseudo-linear accumulation and then eventually stops increasing. Thiseffect appears to be due to non-specific interactions between the DNApolymerase and the double stranded products themselves. The molar ratioof product to enzyme in the plateau phase is typically consistent forseveral DNA polymerases, even when different amounts of enzyme areincluded in the reaction, and is approximately 30:1 product:enzyme. Thiseffect thus limits the total amount of double-stranded product that canbe generated in a PCR reaction such that the number of different lociamplified must be balanced against the total amount of each amplicondesired for subsequent analysis, e.g. by gel electrophoresis, primerextension, etc.

Because of these and other considerations, although multiplexed PCRincluding 50 loci has been reported (Lindblad-Toh et al., Nature Genet.4: 381 (2000)), multiplexing is typically limited to fewer than tendistinct products. However, given the need to analyze as many as 100,000to 450,000 SNPs from a single genomic DNA sample there is a clear needfor a means of expanding the multiplexing capabilities of PCR reactions.

The present invention provides methods for substantial multiplexing ofPCR reactions by, for example, combining the INVADER assay withmultiplex PCR amplification. The INVADER assay provides a detection stepand signal amplification that allows very large numbers of targets to bedetected in a multiplex reaction. As desired, hundreds to thousands tohundreds of thousands of targets may be detected in a multiplexreaction.

Direct genotyping by the INVADER assay typically uses from 5 to 100 ngof human genomic DNA per SNP, depending on detection platform. For asmall number of assays, the reactions can be performed directly withgenomic DNA without target pre-amplification, however, for highlymultiplex reactions, the amount of sample DNA may become a limitingfactor.

Because the INVADER assay provides from 10⁶ to 10⁷ fold amplification ofsignal, multiplexed PCR in combination with the INVADER assay would useonly limited target amplification as compared to a typical PCR.Consequently, low target amplification level alleviates interferencebetween individual reactions in the mixture and reduces the inhibitionof PCR by it's the accumulation of its products, thus providing for moreextensive multiplexing. Additionally, it is contemplated that lowamplification levels decrease a probability of targetcross-contamination and decrease the number of PCR-induced mutations.

Uneven amplification of different loci presents one of the biggestchallenges in the development of multiplexed PCR. Differences inamplification factors between two loci may result in a situation wherethe signal generated by an INVADER reaction with a slow-amplifying locusis below the limit of detection of the assay, while the signal from afast-amplifying locus is beyond the saturation level of the assay. Thisproblem can be addressed in several ways. In some embodiments, theINVADER reactions can be read at different time points, e.g., inreal-time, thus significantly extending the dynamic range of thedetection. In other embodiments, multiplex PCR can be performed underconditions that allow different loci to reach more similar levels ofamplification. For example, primer concentrations can be limited,thereby allowing each locus to reach a more uniform level ofamplification. In yet other embodiments, concentrations of PCR primerscan be adjusted to balance amplification factors of different loci.

The present invention provides for the design and characteristics ofhighly multiplex PCR including hundreds to thousands of products in asingle reaction. For example, the target pre-amplification provided byhundred-plex PCR reduces the amount of human genomic DNA required forINVADER-based SNP genotyping to less than 0.1 ng per assay. Thespecifics of highly multiplex PCR optimization and a computer programfor the primer design are described in U.S. patent application Ser. Nos.10/967,711 and 10/321,039 herein incorporated by reference in theirentireties.

In addition to providing methods for highly multiplex PCR, the presentinvention further provides methods of conducting reverse transcriptionand target and signal amplification reactions in a single reactionvessel with no subsequent manipulations or reagent additions beyondinitial reaction set-up. Such combined reactions are suitable forquantitative analysis of limiting target quantities in very shortreaction times. Methods for conducting such reactions are described inU.S. patent application Ser. No. 11/266,723, herein incorporated byreference in its entirety.

ii. Other Detection Assays

The present invention is not limited to detection of target sequences byINVADER assay. The methods and compositions of the present inventionfind use in increasing the dynamic range of any number detection assaysincluding, but not limited to, those described below.

In some embodiments, the methods and compositions of the presentinvention find use in increasing the dynamic range of a hybridizationassay. A variety of hybridization assays using a variety of technologiesfor hybridization and detection are available. A description of aselection of assays is provided below.

In some embodiments, hybridization of a probe to the sequence ofinterest is detected directly by visualizing a bound probe (e.g., aNorthern or Southern assay; See e.g., Ausabel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY [1991]). In athese assays, genomic DNA (Southern) or RNA (Northern) is isolated froma subject. The DNA or RNA is then cleaved with a series of restrictionenzymes that cleave infrequently in the genome and not near any of themarkers being assayed. The DNA or RNA is then separated (e.g., on anagarose gel) and transferred to a membrane. A labeled (e.g., byincorporating a radionucleotide) probe or probes specific for the targetsequence being detected is allowed to contact the membrane under acondition or low, medium, or high stringency conditions. Unbound probeis removed and the presence of binding is detected by visualizing thelabeled probe.

In some embodiments of the present invention, variant sequences aredetected using a DNA chip (e.g., array) hybridization assay. In thisassay, a series of oligonucleotide probes are affixed to a solidsupport. In some embodiments, the oligonucleotide probes are designed tobe unique to a given target sequence. In preferred embodiments, thearrays comprise multiple probes (e.g., mismatch or different amounts ofa completely complementary probe) in order to increase the dynamic rangeof the assay. The DNA sample of interest is contacted with the DNA“chip” and hybridization is detected.

In some embodiments, the DNA chip assay is a GeneChip (Affymetrix, SantaClara, Calif.; See e.g., U.S. Pat. Nos. 6,045,996; 5,925,525; and5,858,659; each of which is herein incorporated by reference) assay. TheGeneChip technology uses miniaturized, high-density arrays ofoligonucleotide probes affixed to a “chip.” Probe arrays aremanufactured by Affymetrix's light-directed chemical synthesis process,which combines solid-phase chemical synthesis with photolithographicfabrication techniques employed in the semiconductor industry. Using aseries of photolithographic masks to define chip exposure sites,followed by specific chemical synthesis steps, the process constructshigh-density arrays of oligonucleotides, with each probe in a predefinedposition in the array. Multiple probe arrays are synthesizedsimultaneously on a large glass wafer. The wafers are then diced, andindividual probe arrays are packaged in injection-molded plasticcartridges, which protect them from the environment and serve aschambers for hybridization.

The nucleic acid to be analyzed is isolated, amplified by PCR, andlabeled with a fluorescent reporter group. The labeled DNA is thenincubated with the array using a fluidics station. The array is theninserted into the scanner, where patterns of hybridization are detected.The hybridization data are collected as light emitted from thefluorescent reporter groups already incorporated into the target, whichis bound to the probe array. Probes that perfectly match the targetgenerally produce stronger signals than those that have mismatches.Since the sequence and position of each probe on the array are known, bycomplementarity, the identity of the target nucleic acid applied to theprobe array can be determined.

In other embodiments, a DNA microchip containing electronically capturedprobes (Nanogen, San Diego, Calif.) is utilized (See e.g., U.S. Pat.Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are hereinincorporated by reference). Through the use of microelectronics,Nanogen's technology enables the active movement and concentration ofcharged molecules to and from designated test sites on its semiconductormicrochip. DNA capture probes unique to a given SNP or mutation areelectronically placed at, or “addressed” to, specific sites on themicrochip. Since DNA has a strong negative charge, it can beelectronically moved to an area of positive charge.

First, a test site or a row of test sites on the microchip iselectronically activated with a positive charge. Next, a solutioncontaining the DNA probes is introduced onto the microchip. Thenegatively charged probes rapidly move to the positively charged sites,where they concentrate and are chemically bound to a site on themicrochip. The microchip is then washed and another solution of distinctDNA probes is added until the array of specifically bound DNA probes iscomplete.

A test sample is then analyzed for the presence of target DNA moleculesby determining which of the DNA capture probes hybridize, withcomplementary DNA in the test sample (e.g., a PCR amplified gene ofinterest). An electronic charge is also used to move and concentratetarget molecules to one or more test sites on the microchip. Theelectronic concentration of sample DNA at each test site promotes rapidhybridization of sample DNA with complementary capture probes(hybridization may occur in minutes). To remove any unbound ornonspecifically bound DNA from each site, the polarity or charge of thesite is reversed to negative, thereby forcing any unbound ornonspecifically bound DNA back into solution away from the captureprobes. A laser-based fluorescence scanner is used to detect binding,

In still further embodiments, an array technology based upon thesegregation of fluids on a flat surface (chip) by differences in surfacetension (ProtoGene, Palo Alto, Calif.) is utilized (See e.g., U.S. Pat.Nos. 6,001,311; 5,985,551; and 5,474,796; each of which is hereinincorporated by reference). Protogene's technology is based on the factthat fluids can be segregated on a flat surface by differences insurface tension that have been imparted by chemical coatings. Once sosegregated, oligonucleotide probes are synthesized directly on the chipby ink-jet printing of reagents. The array with its reaction sitesdefined by surface tension is mounted on a X/Y translation stage under aset of four piezoelectric nozzles, one for each of the four standard DNAbases. The translation stage moves along each of the rows of the arrayand the appropriate reagent is delivered to each of the reaction site.For example, the A amidite is delivered only to the sites where amiditeA is to be coupled during that synthesis step and so on. Common reagentsand washes are delivered by flooding the entire surface and thenremoving them by spinning.

DNA probes unique for the target nucleic acid are affixed to the chipusing Protogene's technology. The chip is then contacted with thePCR-amplified genes of interest. Following hybridization, unbound DNA isremoved and hybridization is detected using any suitable method (e.g.,by fluorescence de-quenching of an incorporated fluorescent group).

In yet other embodiments, a “bead array” is used for the detection ofpolymorphisms (Illumina, San Diego, Calif.; See e.g., PCT PublicationsWO 99/67641 and WO 00/39587, each of which is herein incorporated byreference). Illumina uses a BEAD ARRAY technology that combines fiberoptic bundles and beads that self-assemble into an array. Each fiberoptic bundle contains thousands to millions of individual fibersdepending on the diameter of the bundle. The beads are coated with anoligonucleotide specific for the detection of a given target nucleiacid. Batches of beads are combined to form a pool specific to thearray. To perform an assay, the BEAD ARRAY is contacted with a preparedsubject sample (e.g., DNA). Hybridization is detected using any suitablemethod.

In other embodiments, the array methods described in U.S. Pat. Nos.6,410,229 and 6,344,316; each of which is incorporated by referenceherein, are utilized.

In some embodiments, hybridization of a bound probe is detected using aTaqMan assay (PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat.Nos. 5,962,233 and 5,538,848, each of which is herein incorporated byreference). The assay is performed during a PCR reaction. The TaqManassay exploits the 5′-3′ exonuclease activity of DNA polymerases such asAMPLITAQ DNA polymerase. A probe, specific for a given allele ormutation, is included in the PCR reaction. The probe consists of anoligonucleotide with a 5′-reporter dye (e.g., a fluorescent dye) and a3′-quencher dye. During PCR, if the probe is bound to its target, the5′-3′ nucleolytic activity of the AMPLITAQ polymerase cleaves the probebetween the reporter and the quencher dye. The separation of thereporter dye from the quencher dye results in an increase offluorescence. The signal accumulates with each cycle of PCR and can bemonitored with a fluorimeter.

In still further embodiments, polymorphisms are detected using theSNP-IT primer extension assay (Orchid Biosciences, Princeton, N.J.; Seee.g., U.S. Pat. Nos. 5,952,174 and 5,919,626, each of which is hereinincorporated by reference). In this assay, SNPs are identified by usinga specially synthesized DNA primer and a DNA polymerase to selectivelyextend the DNA chain by one base at the suspected SNP location. DNA inthe region of interest is amplified and denatured. Polymerase reactionsare then performed using miniaturized systems called microfluidics.Detection is accomplished by adding a label to the nucleotide suspectedof being at the SNP or mutation location. Incorporation of the labelinto the DNA can be detected by any suitable method (e.g., if thenucleotide contains a biotin label, detection is via a fluorescentlylabelled antibody specific for biotin).

In yet other embodiments, the methods and compositions of the presentinvention are utilized with the method described in U.S. Pat. No.6,528,254 (herein incorporated by reference in its entirety). The methodcomprises generating a cleavage structure using a primer and a nucleicacid polymerase and cleaving the cleavage structure with a FENendonuclease.

In other embodiments, a ligase based detection assay is utilized withthe methods and compositions of the present invention. For example, insome embodiments, the method described in U.S. Pat. Nos. 5,521,065 and5,514,543 (each of which is herein incorporated by reference in itsentirety) is utilized. Briefly, the method involves reacting a mixtureof single-stranded nucleic acid fragments with a first probe which iscomplementary to a first region of the target sequence, and with asecond probe which is complementary to a second region of the targetsequence, where the first and second target regions are contiguous withone another, under hybridization conditions in which the two probesbecome stably hybridized to their associated target regions. Followinghybridization, any of the first and second probes hybridized tocontiguous first and second target regions are ligated, and the sampleis tested for the presence of expected probe ligation product. Thepresence of ligated product indicates that the target sequence ispresent in the sample. In some embodiments, the ligation reaction isperformed concurrent with a nucleic acid amplification reaction (Seee.g., U.S. Pat. Nos. 6,130,073 and 5,912,148, each of which is hereinincorporated by reference in its entirety).

In some embodiments, the present invention provides microarrays.Microarrays may be utilized with any of the detection assays describedherein. The below discussion describes microarrays in the context ofINVADER and TAQMAN assays. However, one skilled in the art recognizesthat microarrays may be adapted for use with any number of detectionassays.

Microarrays may comprise assay reagents and/or targets attached to orlocated on or near a solid surface (i.e. a microarray spot is formed)such that a detection assay may be performed on the solid surface. Insome preferred embodiments, the microarray spots are generated topossess specific and defined chemical and physical characteristics. Inother embodiments, the microarray may comprise a plurality of reactionchambers (e.g., capillaries), for conducting detection assays. In somesuch embodiments, nucleic acids or other detection assay components areattached to the surface of the reaction chamber. In other embodiments,detection assay components are all in the liquid phase or dried down inthe reaction chamber.

As used herein, the term “microarray-spot” refers to the discreet areaformed on a solid surface, in a layer of non-aqueous liquid in amicrowell, or in a reaction chamber containing a population of detectionassay reagents. A microarray-spot may be formed, for example, on a solidsubstrate (e.g. glass, TEFLON) or in a layer of non-aqueous liquid orother material that is on a solid surface, when a reagent samplecomprising detection assay reagents is applied to the solid surface (orfilm on a solid surface) by a transfer means (e.g. pin spotting tool,inkjet printer, etc.). In preferred embodiments, the solid substrate(e.g. modified as described below) contains microwells and themicroarray-spots are applied in the microwells. In other embodiments,the solid support serves as a platform on which microwells areprinted/created and the necessary reagents are introduced to thesemicrowells and the subsequent reaction(s) take place entirely insolution. Creation of a microwell on a solid support may be accomplishedin a number of ways, including; surface tension, and etching ofhydrophilic pockets (e.g. as described in patent publications assignedto Protogene Corp.). For example, the surface of a support may be coatedwith a hydrophobic layer, and a chemical component, that etches thehydrophobic layer, is then printed on to the support in small volumes(e.g., to generate local changes in the physical or chemical propertiesof the hydrophobic layer). The printing results in an array ofhydrophilic microwells. An array of printed hydrophobic or hydrophilictowers may be employed to create microarrays. A surface of a slide maybe coated with a hydrophobic layer, and then a solution is printed onthe support that creates a hydrophilic layer on top of the hydrophobicsurface. The printing results in an array of hydrophilic towers.Mechanical microwells may be created using physical barriers,+/−chemical barriers. For example, microgrids such as gold grids may beimmobilized on a support, or microwells may be drilled into the support(e.g. as demonstrated by BML). Also, a microarray may be printed on thesupport using hydrophilic ink such as TEFLON. Such arrays arecommercially available through Precision Lab Products, LLC, Middleton,Wis. In yet another variant, data of customer preferences with respectto the format of the detection assay array are stored on a database usedwith components of the invention. This information can be used toautomatically configure products for a particular customer based uponminimal identification information for a customer, e.g. name, accountnumber or password. In some embodiments, the desired reactionscomponents (e.g., target nucleic acids or detection assay components)are spotted or delivered into wells and then taken up into smallreaction chambers such as capillaries. The reaction then occurs withinthe reaction chamber.

Many types of methods may be used for printing of desired reagents intomicroarrays (e.g. microarray spots printed into microwells). In someembodiments, a pin tool is used to load the array (e.g. generate amicroarray spot) mechanically (see, e.g., Shalon, Genome Methods, 6:639[1996], herein incorporated by reference). In other embodiments, ink jettechnology is used to print oligonucleotides onto a solid surface (e.g.,O'Donnelly-Maloney et al., Genetic Analysis: Biomolecular Engineering,13:151 [1996], herein incorporated by reference) in order to create oneor more microarray spots in a well.

Examples of desired reagents for printing into/onto solid supports (e.g.with microwell arrays) include, but are not limited to, molecularreagents, such as INVADER reaction reagents, designed to perform anucleic acid detection assay (e.g., an array of SNP detection assayscould be printed in the wells); and target nucleic acid, such as humangenomic DNA (hgDNA), resulting in an array of different samples. Also,desired reagents may be simultaneously supplied with the etching/coatingreagent or printed into/onto the microwells/towers subsequent to theetching process. For arrays created with mechanical barriers the desiredreagents are, for example, printed into the resulting wells. In someembodiments, the desired reagents may need to be printed in a solutionthat sufficiently coats the microwell and creates a hydrophilic,reaction friendly, environment such as a high protein solution (e.g.BSA, non-fat dry milk). In certain embodiments, the desired reagents mayalso need to be printed in a solution that creates a “coating” over thereagents that immobilizes the reagents, this could be accomplished withthe addition of a high molecular weight carbohydrate such as FICOLL ordextran. In some embodiments, the coating is oil.

Application of the target solution to the microarray (or reactionreagents if the target has been printed down or taken up in a reactionchamber) may be accomplished in a number of ways. For example, the solidsupport may be dipped into a solution containing the target, or byputting the support in a chamber with at least two openings then feedingthe target solution into one of the openings and then pulling thesolution across the surface with a vacuum or allowing it to flow acrossthe surface via capillary action. Examples of devices useful forperforming such methods include, but are not limited to, TECAN—GenePaintsystem, and AutoGenomics AutoGene System. In yet another embodimentspotters commercially available from Virtek Corp. are used to spotvarious detection assays onto plates, slides and the like.

In some embodiments, solutions (e.g. reaction reagents or targetsolutions) are dragged, rolled, or squeegeed across the surface of thesupport. One type of device useful for this type of application is aframed holder that holds the support. At one end of the holder is aroller/squeegee or something similar that would have a channel forloading of the target solution in front of it. The process of moving theroller/squeegee across the surface applies the target solution to themicrowells. At the end opposite end of the holder is a reservoir thatwould capture the unused target solution (thus allowing for reuse onanother array if desired). Behind the roller/squeegee is an evaporationbarrier (e.g., mineral oil, optically clear adhesive tape etc.) and itis applied as the roller/squeegee move across the surface.

The application of a target solution to microwell or reaction chamberarrays results in the deposition of the solution at each of themicrowell or reaction chamber locations. The chemical and/or mechanicalbarriers would maintain the integrity of the array and preventcross-contamination of reagents from element to element. In somepreferred embodiments, materials in the microwells or reaction chambersare dried. In some such embodiments, the reagents are rehydrated by thetarget solution (or detection assay component solution) resulting in anultra-low volume reaction mix. In some embodiments, the microarrayreactions are covered with mineral oil or some other suitableevaporation barrier or humidity chamber to allow high temperatureincubation. The signal generated may be detected directly through theapplied evaporation barrier using a fluorescence microscope, arrayreader or standard fluorescence plate reader.

Advantages of the use of a microwell-microarray, for running INVADERassays (e.g. dried down INVADER assay components in each well) include,but are not limited to: the ability to use the INVADER Biplex format fora nucleic acid detection assay; sufficient sensitivity to detect hgDNAdirectly, the ability to use “universal” FRET cassettes; no attachmentchemistry needed (which means already existing off the shelf reagentscould be used to print the microarrays), no need to fractionate hgDNA toaccount for surface effect on hybridization, low mass of hgDNA needed tomake tens of thousands of calls, low volume need (e.g. a 100 μmmicrowell would have a volume of 0.28 nl, and at 10⁴ microwells perarray a volume of 2.8 μl would fill all wells), a solution of 333 ng/μlhgDNA would result in ˜100 copies per microwell (this is 33× moreconcentrated than the use of 100 ng hgDNA in a 20 μl reaction), thus 2.8μl×333 ng/μl=670 ng hgDNA for 10⁴ calls or 0.07 ng per call. It isappreciated that other detection assays can also be presented in thisformat.

Generating and Using Microarray-Spots with Non-Aqueous Liquids

In certain preferred embodiments, the present invention provides methodsfor generating microarray spots in wells by applying a detection assayreagent solution to a well containing non-aqueous liquid. In otherpreferred embodiments, the present invention provides methods ofcontacting a microarray-spot with a test sample solution (e.g.comprising target nucleic acids) by shooting the test sample solutionthrough a layer of non-aqueous liquid covering the microarray spot. Incertain embodiments, the solid supports are coated with sol-gel films(described below in more detail).

In some embodiments, the present invention provides methods comprising;a) providing; i) a solid support comprising a well, ii) a non-aqueousliquid, and iii) a detection reagent solution; and b) adding thenon-aqueous liquid to the well, and c) adding the detection reagentsolution to the well through the non-aqueous liquid under conditionssuch that at least one microarray-spot is formed in the well. In otherembodiments, the methods further comprise step d) contacting the atleast one microarray-spot with a test sample solution. In additionalembodiments, the contacting comprises propelling the test samplesolution through the non-aqueous liquid in the well.

In particular embodiments, the non-aqueous liquid is oil. In otherembodiments, the solid support comprises a plurality of wells, and themethod is performed with the plurality of wells. In further embodiments,at least two microarray-spots are formed simultaneously (e.g. in atleast two of the plurality of wells).

In some embodiments, the test sample solution comprises a target nucleicacid molecule. In preferred embodiments, the target solution comprisesless than 800 copies of a target nucleic acid molecule, or less than 400copies of a target nucleic acid molecule or less than 200 copies of atarget nucleic acid molecule. In particular embodiments, the contactingthe microarray-spot with the test sample solution identifies thepresence or absence of a polymorphism, or other desired particularsequence to be detected, in the target nucleic acid molecule. In someembodiments, wells are coated with a sol-gel coating (e.g. prior tomicroarray-spot formation).

In other embodiments, the detection reagent solution comprisescomponents configured for use with a detection assay selected from;TAQMAN assay, or an INVADER assay, a polymerase chain reaction assay, arolling circle extension assay, a sequencing assay, a hybridizationassay employing a probe complementary to the polymorphism, a bead arrayassay, a primer extension assay, an enzyme mismatch cleavage assay, abranched hybridization assay, a NASBA assay, a molecular beacon assay, acycling probe assay, a ligase chain reaction assay, and a sandwichhybridization assay. In preferred embodiments, the detection reagentsolution comprises INVADER oligonucleotides, and 5′ probeoligonucleotides.

In additional embodiments, the contacting is performed with a SYNQUADnanovolume pipetting system, or other fluid transfer system or device.In preferred embodiments, the commercially available CARTESIAN SYNQUADnanovolume pipetting system is employed. Similar devices may also beemployed, including those described in U.S. Pat. No. 6,063,339 and U.S.Pat. No. 6,258,103, both of which are specifically incorporated byreference, as well as PCT applications: WO0157254; WO0049959; WO0001798;and WO9942804; all of which are specifically incorporated by reference.

In particular embodiments, at least 2 microarray-spots are formed in thewell (or at least 3 or 4 or 5 microarray-sports are formed in eachwell). In multi-well formats, employing multiple microarray-spotsmultiplies the number of reactions that can be performed on a singlesolid support (e.g. if 4 microarray-spots are formed in each of the 1536wells in an a 1536 well plate, then 6144 microarray-spots would beavailable for performing detection reactions). In further embodiments,the present invention provides a solid support with a well (or wells)formed by the methods described above.

In some embodiments, the present invention provides methods comprising;a) providing; i) a solid support comprising a microarray-spot, ii) anon-aqueous liquid; and iii) a test sample solution; and b) covering themicroarray-spot with a layer of the non-aqueous liquid, and c)contacting the microarray-spot with the test sample solution through thelayer of non-aqueous liquid. In other embodiments, the test samplesolution comprises a target nucleic acid molecule. In furtherembodiments, the contacting identifies the presence or absence of atleast one polymorphism in the target nucleic acid molecule. In preferredembodiments, the test sample solution comprises a target nucleic acidmolecule. In preferred embodiments, the target solution comprises lessthan 800 copies of a target nucleic acid molecule, or less than 400copies of a target nucleic acid molecule or less than 200 copies of atarget nucleic acid molecule.

In certain embodiments, the microarray-spot comprises componentsconfigured for use with a detection assay selected from; TAQMAN assay,or an INVADER assay, a polymerase chain reaction assay, a rolling circleextension assay, a sequencing assay, a hybridization assay employing aprobe complementary to the polymorphism, a bead array assay, a primerextension assay, an enzyme mismatch cleavage assay, a branchedhybridization assay, a NASBA assay, a molecular beacon assay, a cyclingprobe assay, a ligase chain reaction assay, and a sandwich hybridizationassay. In preferred embodiments, the microarray-spot comprises INVADERoligonucleotides, and 5′ probe oligonucleotides.

In some embodiments, the solid support comprises a well, and themicroarray-spot is located in the well. In certain embodiments, thenon-aqueous liquid is oil. In other embodiments, the solid supportcomprises a plurality of wells, and the method is performed with theplurality of wells. In particular embodiments, at least twomicroarray-spots are formed simultaneously. In some embodiments, atleast 2 microarray-spots are formed in the well (or at least 3 or 4 or 5microarray-sports are formed in each well). In multi-well formats,employing multiple microarray-spots multiplies the number of reactionsthat can be performed on a single solid support (e.g. if 4microarray-spots are formed in each of the 1536 wells in an a 1536 wellplate, then 6144 microarray-spots would be available for performingdetection reactions; if etched 3072 well plates are used, additionalspots may be formed). In further embodiments, the present inventionprovides a solid support with a well (or wells) formed by the methodsdescribed above.

In some embodiments, the contacting comprises propelling the test samplesolution through the non-aqueous liquid in the well. In otherembodiments, the non-aqueous liquid is mineral oil. In additionalembodiments, the non-aqueous liquid is selected from mineral oil, a seedoil, and an oil derived from petroleum.

In additional embodiments, the contacting is performed with a SYNQUADnanovolume pipetting system, or other fluid transfer system or device.In preferred embodiments, the commercially available CARTESIAN SYNQUADnanovolume pipetting system is employed. Similar devices may also beemployed, including those described in U.S. Pat. No. 6,063,339 and U.S.Pat. No. 6,258,103, both of which are specifically incorporated byreference, as well as PCT applications: WO0157254; WO0049959; WO0001798;and WO9942804; all of which are specifically incorporated by reference.

In some embodiments, the present invention provides systems comprising;a) a nonvolume pipetting system (e.g., SYNQUAD), and b) a solid supportcomprising a microarray-spot, wherein the microarray spot is coveringwith a layer of a non-aqueous liquid. In other embodiments, the systemfurther comprises a test sample solution.

iv. Formats for Assays on a Solid Support

In some embodiments, detection assays are performed on a solid support.The below discussion describes assays on a solid support in the contextof the INVADER assay. However, one skilled in the relevant artsrecognizes that the methods described herein can be adapted for use withany nucleic acid detection assay (e.g., the detection assays describedherein).

The present invention is not limited to a particular configuration ofthe INVADER assay. Any number of suitable configurations of thecomponent oligonucleotides may be utilized. For example, in someembodiments of the present invention, the probe oligonucleotide is boundto a solid support and the INVADER oligonucleotide and genomic DNA (orRNA) target are provided in solution. In other embodiments of thepresent invention, the INVADER oligonucleotide is bound to the supportand the probe and target are in solution. In yet other embodiments, boththe probe and INVADER oligonucleotides are bound to the solid support.In further embodiments, the target nucleic acid is bound directly orindirectly (e.g., through hybridization to a bound oligonucleotide thatis not part of a cleavage structure) to a solid support, and either orboth of the probe and INVADER oligonucleotides are provided either insolution, or bound to a support. In still further embodiments, a primaryINVADER assay reaction is carried out in solution and one or morecomponents of a secondary reaction are bound to a solid support. In yetother embodiments, all of the components necessary for an INVADER assayreaction, including cleavage agents, are bound to a solid support.

The present invention is not limited to the configurations describedherein. Indeed, one skilled in the art recognizes that any number ofadditional configurations may be utilized. Any configuration thatsupports a detectable invasive cleavage reaction may be utilized.Additional configurations are identified using any suitable method,including, but not limited to, those disclosed herein.

In some embodiments, the probe oligonucleotide is bound to a solidsupport. In some embodiments, the probe is a labeled Signal Probeoligonucleotide. The signal probe is cleaved to release a signalmolecule indicative of the presence of a given target molecule. In someembodiments, the signal molecule is a fluorescence donor in an energytransfer reaction (e.g., FRET), whose emission increases in response toseparation from a quenching fluorescence acceptor. In other embodiments,the signal molecule is a fluorescent moiety that is detected only uponits release into solution. It yet other embodiments, the signal moleculeis a fluorescently labeled small molecule that is separated from thefull length Signal Probe by carrying a distinct charge.

In some embodiments, a system is designed in which no separation stepsare required to visualize the signal generated by the reaction. In someembodiments, this is accomplished in the FRET system in which thefluorescence donor remains affixed to the solid support followingcleavage of the signal probe. This design has several complexities thatstem from the nature of the FRET reaction. The quenching in the FRETsignal molecule is only 97-99% efficient (i.e. not all of the energyemitted by the donor will be absorbed by the quencher). To detect thefluorescence of the unquenched donor above the background of theuncleaved probes, it is necessary to cleave 1-3% of the probe molecules.Assuming that in a 100 μm×100 μm area, there are ˜10⁸ probes bound, then˜10⁶ should be cleaved to generate a signal detectable above theinherent background generated by those probes. Probe cycling in anINVADER assay reaction on a single target molecule can generateapproximately 1000-2000 cleaved probe molecules per hour (assuming aturnover rate of 15-30 events/target/min). Roughly 1000 target moleculesare required to generate this level of cleaved Signal Probes. Assuming areaction volume of 1 nL, the necessary target concentration becomes 1pM, well within the range of the maximum that can be manipulated (e.g.,0.5-2.5 pM). At less than maximal probe densities, it would nonethelessbe necessary to deliver at least 10-20 target molecules (i.e. a 10-20 fMsolution) to each reaction area to ensure a statistical likelihood thateach will contain target. The same target concentration considerationsapply to other, non-FRET alternatives, for example, release of a singlefluorescent group into solution, with or without a quenching fluorophoreand release of a positively charged signal molecule even though <1%cleavage would be detectable with these other methods. Accordingly, insome embodiments, dilute solutions are used in conjunction with longerreaction times (e.g. a 100 fM solution could be applied and thereactions run for 10-24 hours).

In some embodiment of the present invention, the INVADER oligonucleotideis bound to the solid support and the probe oligonucleotide is free insolution. In this embodiment, there are no restrictions on the length ofthe INVADER oligonucleotide-target duplex, since the INVADERoligonucleotide does not need to cycle on and off the target, as doesthe signal probe. Thus, in some embodiments where the INVADERoligonucleotide is bound to a solid support, the INVADER oligonucleotideis used as a “capture” oligonucleotide to concentrate target moleculesfrom solution onto the solid phase through continuous application ofsample to the solid support. For example, by applying 1 ml of a 1 mg/mltarget solution, it is possible to bind 10⁶-10⁸ target molecules in a100 μM×100 μM area. Moreover, because the INVADER oligonucleotide-targetinteraction is designed to be stable, in some embodiments, the supportis washed to remove unbound target and unwanted sample impurities priorto applying the signal probes, enzyme, etc., to ensure even lowerbackground levels. In other embodiments, a capture oligonucleotidecomplementary to a distinct region in the proximity of the locus beinginvestigated is utilized.

Several possibilities exist for separation of cleaved from uncleavedsignal probe reactions where INVADER oligonucleotides are bound thesolid support and signal probe oligonucleotides are free in solution. Inpreferred embodiments, a labeling strategy is utilized that makes itpossible to chemically differentiate cleaved from uncleaved probe sinceboth full length and cleaved probes are in solution. For example, insome embodiments (e.g., FRET signal probe), full length probe isquenched but the cleavage product generates fluorescent signal. In otherembodiment (e.g, CRE), the full length probe is negatively charged butthe cleaved probe is positively charged.

However, in some preferred embodiments, CRE separation is utilized.First, the cleaved signal probes generated by the CRE approach areactively captured on a negatively charged electrode. This captureresults in partitioning from uncleaved molecules as well asconcentration of the labeled, cleaved probes by as much as an order ofmagnitude. Second, the use of an electric field to capture the cleavedprobe eliminates the need to micromachine tiny wells to preventdiffusion of the cleaved probes.

In some embodiments of the present invention, both a probe and anINVADER oligonucleotide are bound to a solid support. In preferredembodiments, probe and INVADER oligonucleotides are placed in closeproximity on the same solid support such that a target nucleic acid maybind both the probe and INVADER oligonucleotides. In some embodiments,the oligonucleotides are attached via spacer molecules in order toimprove their accessibility and decrease interactions betweenoligonucleotides.

In some preferred embodiments, a single INVADER oligonucleotide isconfigured to allow it to contact and initiate multiple cleavagereactions. For example, in some embodiments, one INVADER oligonucleotideis surrounded on a solid support by multiple Signal Probeoligonucleotides. A target nucleic acid binds to an INVADER and a probeoligonucleotide. The Signal Probe is cleaved (generating signal) andreleased, leaving the target bound to the INVADER oligonucleotide. Thistarget:INVADER oligonucleotide complex is then able to contact anotherSignal Probe and promote another cleavage event. In this manner, thesignal generated from one target and one INVADER oligonucleotide isamplified.

In other embodiments, the probe and INVADER oligonucleotides arecombined in one molecule. The connection between the probe and INVADERportions of the single molecule may be nucleic acid, or may be anon-nucleic acid linker (e.g., a carbon linker, a peptide chain).

In some embodiments, a primary INVADER assay reaction is performed insolution and a secondary reaction is performed on a solid support.Cleaved probes from the primary INVADER assay reaction are contactedwith a solid support containing one or more components of a cleavagestructure, including but not limited to a secondary target nucleic acid,a secondary probe or a secondary INVADER oligonucleotide. In a preferredembodiment, the component is a one-piece secondary oligonucleotide, orcassette, comprising both a secondary target portion and a secondaryprobe portion. In a particularly preferred embodiment, the cassette islabeled to allow detection of cleavage of the cassette by a FRET. Thesecondary signal oligonucleotide may be labeled using any suitablemethod including, but not limited to, those disclosed herein. It will beappreciated that any of the embodiments described above for configuringan INVADER assay reaction on a support may be used in configuring asecondary or subsequent INVADER assay reaction on a support.

In some embodiments of the present invention, the target nucleic acid(e.g, genomic DNA) is bound to the solid support. In some embodiments,the INVADER and Probe oligonucleotides are free in solution. In otherembodiments, both the target nucleic acid, the INVADER oligonucleotide,and the Probe (e.g, Signal Probe) oligonucleotides are bound. In yetother embodiments, a secondary oligonucleotide (e.g, a FREToligonucleotide) is included in the reaction. In some embodiments, theFRET oligonucleotide is free in solution. In other embodiments, the FREToligonucleotide is bound to the solid support.

In some embodiments, the cleavage means (e.g., enzyme) is bound to asolid support. In some embodiments, the target nucleic acid, probeoligonucleotide, and INVADER oligonucleotide are provided in solution.In other embodiments, one or more of the nucleic acids is bound to thesolid support. Any suitable method may be used for the attachment of acleavage enzyme to a solid support, including, but not limited to,covalent attachment to a support (See e.g., Chemukhin and Klenova, Anal.Biochem., 280:178 [2000]), biotinylation of the enzyme and attachmentvia avidin (See e.g., Suter et al., Immunol. Lett. 13:313 [1986]), andattachment via antibodies (See e.g., Bilkova et al., J. Chromatogr. A,852:141 [1999]).

In some embodiments of the present invention, oligonucleotides areattached to a solid support via a spacer or linker molecule. The presentinvention is not limited to any one mechanism. Indeed, an understandingof the mechanism is not necessary to practice the present invention.Nonetheless, it is contemplated that spacer molecules enhance INVADERassay reactions by improving the accessibility of oligonucleotides anddecreasing interactions between oligonucleotides. The use of linkers,which can be incorporated during oligonucleotide synthesis, has beenshown to increase hybridization efficiency relative to captureoligonucleotides that contain no linkers (Guo et al., Nucleic AcidsRes., 22:5456 [1994]; Maskos and Southern, Nucleic Acids Res., 20:1679[1992]; Shchepinov et al., Nucleic Acids Research 25:1155 [1997]).

Spacer molecules may be comprised of any suitable material. Preferredmaterials are those that are stable under reaction conditions utilizedand non-reactive with the components of the INVADER assay. Suitablematerials include, but are not limited to, carbon chains (e.g.,including but not limited to C₁₈), poly nucleotides (e.g., including,but not limited to, polyI, polyT, polyG, polyC, and polyA), andpolyglycols (e.g., hexaethylene glycol).

Spacer molecules may be of any length. Accordingly in some embodiments,multiple spacer molecules are attached end to end to achieve the desiredlength spacer. For example, in some embodiments, multiple C₁₈ orhexaethylene glycol spacers (e.g., including, but not limited to, 5, 10,or 20 spacer molecules) are combined. The optimum spacer length isdependent on the particular application and solid support used. Todetermine the appropriate length, different lengths are selected (e.g,5, 10, or 20 C₁₈ or hexaethylene glycol spacers molecules) and reactionsare performed as described herein to determine which spacer gives themost efficient reaction.

The present invention is not limited to any one solid support. In someembodiments, reactions are performed on microtiter plates (e.g.,polystyrene plates containing either containing 96 or 384 wells). Forexample, in some embodiments, streptavidin (SA) coated 96-well or384-well microtiter plates (Boehringer Mannheim Biochemicals,Indianapolis, Ind.) are used as solid supports. In such embodiments,signal can be measured using standard fluorescent, chemiluminescent orcolorimetric microtiter plate readers.

In some embodiments, INVADER assay reactions are carried out onparticles or beads. The particles can be made of any suitable material,including, but not limited to, latex. In some embodiments, columnscontaining a particle matrix suitable for attachment of oligonucleotidesare used. In a some embodiments, reactions are performed in minicolumns(e.g. DARAS, Tepnel, Cheshire, England). The columns contain microbeadsto which oligonucleotides are covalently bound and subsequently used ascapture probes or in enzymatic reactions. The use of minicolumns allowsapproximation of the bound oligonucleotide concentrations that will beattainable in a miniaturized chip format. Oligonucleotide binding islimited by the capacity of the support (i.e. ˜10¹²/cm²). Thus, boundoligonucleotide concentration can only be increased by increasing thesurface area to volume ratio of the reaction vessel. For example, onewell of a 96-well microtiter plate, with a surface area of ˜1 cm² and avolume of 400 μl has a maximal bound oligonucleotide concentration of˜25 nM. On the other hand, a 100 μm×100 μm×100 μM volume in a microchiphas a surface area of 10⁴ μm² and a volume of 1 nL, resulting in a boundoligonucleotide concentration of 0.2 μM. Similar increased surface area:volume ratios can be obtained by using microbeads. Given a bindingcapacity of ≧10¹⁴ oligonucleotides in a 30 μl volume, these beads allowbound oligonucleotide concentrations of 0.2-10 μM, i.e. comparable tothose anticipated for microchips.

In some embodiments, INVADER reaction are carried out on a HydroGel(Packard Instrument Company, Meriden, Conn.) support. HydroGel is porous3D hydrophilic polymer matrix. The matrix consists of a film ofpolyacrylamide polymerized onto a microscope slide. A coupling moiety isco-polymerized into the matrix that permits the immobilization ofaminated oligonucleotide molecules by reductive amination. Covalentattachment by amine groups permits the immobilization of nucleic acidprobes at specific attachment points (usually their ends), and thehydrogel provides a 3D matrix approximating a bulk solution phase,avoiding a solid/solution phase interface.

In other embodiments, INVADER reactions are conducted on a solid supportusing a BEADARRAY (Illumina, San Diego, Calif.) technology. Thetechnology combines fiber optic bundles and beads that self-assembleinto an array. Each fiber optic bundle contains thousands to millions ofindividual fibers depending on the diameter of the bundle. Sensors areaffixed to each beads in a given batch. The particular molecules on abead define that bead's function as a sensor. To form an array, fiberoptic bundles are dipped into pools of coated beads. The coated beadsare drawn into the wells, one bead per well, on the end of each fiber inthe bundle.

The present invention is not limited to the solid supports describedabove. Indeed, a variety of other solid supports are contemplatedincluding, but not limited to, glass microscope slides, glass wafers,gold, silicon, microchips, and other plastic, metal, ceramic, orbiological surfaces.

In some embodiments of the present invention, solid supports are coatedwith a material to aid in the attachment of oligonucleotides. Thepresent invention is not limited to any one surface coating. Indeed, avariety of coatings are contemplated including, but not limited to,those described below.

In some embodiments, solid support INVADER assay reactions are carriedout on solid supports coated with gold. The gold can be attached to anysuitable solid support including, but not limited to, microparticles,microbeads, microscope slides, and microtiter plates. In someembodiments, the gold is functionalized with thiol-reactive maleimidemoieties that can be reacted with thiol modified DNA (See e.g., Frutoset al., Nuc. Acid. Res., 25:4748 [1997]; Frey and Corn, Analytical Chem,68:3187 [1996]; Jordan et al., Analytical Chem, 694939 [1997]; and U.S.Pat. No. 5,472,881; herein incorporated by reference).

In other embodiments, solid support INVADER assay reactions are carriedout on supports coated with silicon. The silicon can be attached to anysuitable support, including, but not limited to, those described aboveand in the illustrative examples provided below.

Additionally, in some embodiments, solid supports are coated with amolecule (e.g., a protein) to aid in the attachment of nucleic acids.The present invention is not limited to any particular surface coating.Any suitable material may be utilized including, but not limited to,proteins such as streptavidin. Thus, in some embodiments,oligonucleotides are attached to solid supports via terminal biotin orNH₂-mediated linkages included during oligonucleotide synthesis. INVADERoligonucleotides are attached to the support at their 5′ ends and SignalProbes are attached at their 3′ ends. In some embodiment,oligonucleotides are attached via a linker proximal to the attachmentpoint. In a preferred embodiment, attachment is via a 40 atom linkerwith a low negative charge density as described in (Shchepinov et al.,Nucleic Acids Research 25: 1155 [1997]).

In other embodiments, oligonucleotides are attached to solid support viaantigen: antibody interaction. For Example, in some embodiments, anantigen (e.g., protein A or Protein G) is attached to a solid supportand IgG is attached to oligonucleotides. In other embodiments, IgG isattached to a solid support and an antigen (e.g., Protein A or ProteinG) is attached to oligonucleotides.

In some embodiments, oligonucleotides are targeted to specific sites onthe solid support. As noted above, when multiple oligonucleotides arebound to the solid support, the oligonucleotides may be synthesizeddirectly on the surface using any number of methods known in the art(e.g., including but not limited to methods described in PCTpublications WO 95/11995, WO 99/42813 and WO 02/04597, and U.S. Pat.Nos. 5,424,186; 5,744,305; and 6,375,903, each incorporated by referenceherein).

Any number of techniques for the addressing of oligonucleotides may beutilized. For example, in some embodiments, solid support surfaces areelectrically polarized at one given site in order to attract aparticular DNA molecule (e.g, Nanogen, CA). In other embodiments, a pintool may be used to load the array mechanically (Shalon, Genome Methods,6:639 [1996]. In other embodiments, ink jet technology is used to printoligonucleotides onto an active surface (e.g., O'Donnelly-Maloney etal., Genetic Analysis: Biomolecular Engineering, 13:151 [1996]).

In some preferred embodiments utilizing gold surfaces, the gold surfacesare further modified to create addressable DNA arrays by photopatterningself-assembled monolayers to form hydrophilic and hydrophobic regions.Alkanethiol chemistry is utilized to create self-assembled monolayers(Nuzzo et al., JACS, 105:4481 [1983]). DNA is placed on the hydrophilicregions by using an automated robotic device (e.g., a pin-loading tool).

v. Reaction Vessels

The detection assays of the present invention may be performed using anysuitable reaction vessel. As used herein, the term “reaction vessel”refers to a system in which a reaction may be conducted, including butnot limited to test tubes, wells, microwells (e.g., wells in microtitreassay plates such as, 96-well, 384-well and 1536-well assay plates),capillary tubes, ends of fibers such as optical fibers, microfluidicdevices such as fluidic chips, cartridges and cards (including but notlimited to those described, e.g., in U.S. Pat. No. 6,126,899, toWoudenberg, et al., U.S. Pat. Nos. 6,627,159, 6,720,187, and 6,734,401to Bedingham, et al., U.S. Pat. Nos. 6,319,469 and 6,709,869 to Mian, etal., U.S. Pat. Nos. 5,587,128 and 6,660,517 to Wilding, et al.), or atest site on any surface (including but not limited to a glass, plasticor silicon surface, a bead, a microchip, or an non-solid surface, suchas a gel or a dendrimer).

In some preferred embodiments, reactions are conducted using a 3Mmicrofluidic card (3M, St. Paul, Minn.). The 3M card has 8 loadingports, each of which is configured to supply liquid reagent to 48individual reaction chambers upon centrifugation of the card. Thereaction chambers contain pre-dispensed and dried assay reactioncomponents for detection of target nucleic acids. These reagents aredissolved when they come in contact with the liquid reagents uponcentrifugation of the card.

III. T-Structure Invasive Cleavage Assays and Amplification Methods

In certain embodiments, the present invention provides methods, kits,and compositions for performing invasive cleavage assays (e.g., theINVADER assay) in a T-structure configuration, where the T-structure isformed by the combination of a stem oligonucleotide, an upstreamoligonucleotide, and a downstream probe. These oligonucleotides may beprovided in a reaction or formed in the reaction (e.g., bypolymerization). An exemplary embodiment of a T-structure invasivecleavage assay, specifically employing the INVADER assay, is shown inFIG. 6. As shown in FIG. 6, T-structure assays involve the use of a stemoligonucleotide that has: 1) a 3′ target specific region configured tohybridize to a target sequence, and 2) a stem region which is configuredto form the target region for an invasive cleavage assay, such as theINVADER assay. Also as shown in FIG. 6, the upstream oligonucleotide hasthree regions: 1) a 5′ target specific region configured to hybridize tothe target sequence; 2) a stem specific region configured to hybridizeto the stem region of the stem oligonucleotide, and 3) a 3′ regionconfigured to not hybridize to the stem region. The thirdoligonucleotide used to form a T-structure for invasive cleavage assaysis the downstream probe, which has: 1) a 3′ region configured tohybridize to the stem region of the stem oligonucleotide and 2) a 5′region configured to not hybridize to the stem region of the stemoligonucleotide.

Generally, the region of inter-oligonucleotide complimentarity betweenthe stem oligonucleotide and upstream oligonucleotide is sufficientlyshort (e.g. FIG. 6 shows a 9 base region) such that stable hybridizationof the two is not favored at the temperature of the invasive cleavageassay employed (e.g. INVADER assay). However, in the presence of thetarget, as shown in FIG. 6, the two oligonucleotides are brought totogether to form a secondary structure. It is noted that the targetspecific regions of the stem and upstream oligonucleotides arepreferably contiguous on the target sequence. However, in certainembodiments, there is a gap between the two target specific regions(e.g. one base gap, two base gap, three base gap, etc.).

When the downstream probe is added with sequence complimentary toadditional non-target sequence in the stem oligonucleotide, an invasivecleavage structure is formed and recognized by a DNA CLEAVASE enzyme.FIG. 6A shows a boxed area that can be recognized by a cleavage agentsuch that the downstream probe is cleaved, resulting in the cleavage ofthe downstream probe as shown in FIG. 6B. FIG. 6B shows the exemplarycleaved portion that is generated, that can then be detected by, forexample, serving as the upstream oligonucleotide in a secondary invasivecleavage reaction that releases a detectable signal (e.g., as shown inFIG. 5).

One advantage of the T-structure invasive cleavage assays is the abilityto use a single cleavage enzymes when detecting RNA templates (e.g.viral RNA targets) in multiplex reactions containing DNA targets or whenuse of hairpin FRET cassettes is desired. Generally, an enzyme optimalfor detection of invasive cleavage structure forming DNA oligos on anRNA template is different from the enzyme optimal for secondarydetection of cleaved DNA flaps on a DNA FRET-probe template. T-structureinvasive cleavage assays allows the use of a single cleavage agent thatis optimized for use with DNA targets since no cleavage structuresinvolving RNA are involved, even thought the original target sequencemay be RNA.

The T-structure invasive cleavage assays of the present invention haveadditional advantages. Because the binding site for the downstream probeoligonucleotide is on the user-supplied stem oligonucleotide, multiplestem oligonucleotides having different target specific regions and thesame stem regions can be used simultaneously to detect multipledifferent regions of the same template (e.g., multiple sites on the sameviral RNA genome). The primary cleavage reaction will generate manycopies of the same cleaved flap for detection in a single secondaryinvasive cleavage reaction (e.g. as shown in FIG. 5). In this way, thespecificity and sensitivity of the system can be increased as the signalgenerated in the presence of template increases non-linearly with thenumber of different T-structure forming oligonucleotide sets used pergiven number of template copies. The detection of the formation andcleavage of the cleavage structure can be detected both directly (e.g.detecting the cleaved flaps) or indirectly (e.g., measuring some otherpart of the assay indicative of the formation and cleavage of thecleavage structure).

The use of T-structures invasive cleavage assays is not limited to thedetection of RNA templates, but can be used for DNA templates as well.As noted above, several T-structure forming oligonucleotide sets withthe same stem region can be used simultaneously to increase thespecificity and/or sensitivity of the assay. Other embodiments includethe use of third, fourth, fifth, and more oligonucleotides to form morecomplicated superstructures. These multiple oligonucleotide structurescan be designed so that only two of the set contain regions with bindingaffinity for a target nucleotide, and the remaining oligonucleotidescontain affinity for each other to form higher-order structures on thetemplate DNA such as cruciform of X-structures, star structures, andothers. These embodiments can increase the sensitivity, specificity, andstability of the detection assay. In an additional embodiment,oligonucleotides containing modified bases (such as 2′-O-methylation, orthe like) can be used to increase the specificity, sensitivity, ordynamic range of the detection assay.

In other embodiments, the T-structure cleavage assays are combined withDNA polymerization. In certain embodiments, two T-structureoligonucleotides are designed to anneal to a target template and to eachother to form a stem region composed of two annealed oligonucleotides ofunequal length, such that the shorter of the two has a 3′ end availablefor the addition of nucleotide bases by a nucleic acid polymerase, usingthe longer of the two stem forming oligos as the template for theextension reaction. This polymerization will create a new hybrid DNAmolecule called an extended upstream oligonucleotide as shown in FIG.7A. This new DNA molecule may then be detected directly by an invasivecleavage assay, such as the INVADER assay, or amplified to produceadditional copies in order to increase the sensitivity and/orsensitivity of the detection assay (see FIG. 7B). As seen in FIG. 7B, aprimer can be added that is complimentary to the newly created upstreamoligo extended region. This primer can be extended as shown in FIG. 7Bto create a stem amplicon sequence. In the presence of a plurality ofprimers and plural of upstream oligonucleotides, PCR could be conductedwith the extended upstream oligonucleotide and stem amplicon serving astemplates. The PCR process, or similar amplification process, could beconducted until the desired amount of stem amplicons and/or extendedupstream oligonucleotides are created.

As can be seen in FIG. 7C, the stem amplicon sequence can act in thesame manner as the stem oligonucleotide and serve as the target forforming an invasive cleavage reaction with a downstream probe andupstream oligonucleotide. The combination of PCR and invasive cleavagedetection allows low levels of target nucleic acid to be detected. Ofcourse, other methods besides invasive cleavage reactions can be used todetect the formation of stem amplicon sequences (e.g. radioactive baseincorporation).

In one embodiment, oligonucleotide primers complimentary to two regionswithin the newly created extended upstream oligonucleotide can be usedto create multiple copies of the stem region sequence, which can then bedetected by an invasive cleavage assay. In some embodiments, theT-structure oligonucleotides, PCR primers, and invasive cleavage assayoligonucleotides are designed in such a way as to detect the oppositestrand of the hybrid oligonucleotide that would be created in the firstand subsequent cycles of PCR. This embodiment improves the specificityand sensitivity of the assay be demanding that both the first extensionstep, and at least one-cycle of sequence specific PCR occur to createthe template that will be detected by the invasive cleavage assay. Asabove, this system may be further improved through the use ofmultiplexing several T-structure forming oligonucleotides, PCR primers,and invasive cleavage assays. Additional methods and compositions usefulfor combination with the T-structure invasive cleavage structure assaysof the present invention are found in U.S. Pat. Nos. 5,424,413 and5,451,503, both of which are herein incorporated by reference in theirentireties as if fully set forth herein.

IV. Nucleic Acid Dispensing Using Non-Ionic Detergents

The present invention also provides methods, compositions, devices, andsystems for consistent nucleic acid dispensing using non-ionicdetergents. It has been found that the use of non-ionic detergents innucleic acid mixtures improves the performance hydrophobic polymerdispensing tips and allows for improved accuracy and reproducibility,for example, in high throughput manufacture of microfluidic sampleprocessing devices. In certain embodiments, the use of detergents inmanufacturing solutions dramatically improves accuracy and increasesmanufacturing throughput where oligonucleotide assay components aredisposed to components of microfluidic devices during manufacturing.

Increasing throughput of biological or chemical assays reduces reagentuse, costs, and result waiting times. Often, assay throughput isincreased through the use of multi-well plates or microfluidic deviceswith multiple process chambers. In some instances, microfluidic deviceswill be configured so as to enable one sample to be preciselydistributed to a plurality of different process chambers where aseparate assay or replicate assays may be performed simultaneously.

Manufacturing methods to improve such microfluidic devices often attemptto dispose one or more components of an assay into different regions ofthe device during manufacturing, so fewer assay components need to beadded by the end user. In particular, when one sample is going to bedistributed to multiple process chambers that must not becomecross-contaminated with each other later, it is advantageous to disposecertain assay components directly into their process chamber locationduring the manufacture and assembly of the microfluidic device, ratherthan distribute them through the internal fluidic connections of themicrofluidic device.

A common type of biological assay adapted to microfluidic devices is anoligonucleotide assay. These assays are useful for the detection of DNAor RNA, or other nucleic acids, through a variety of detectionprotocols, including PCR, OLA, TMA, INVADER Assay, and the like. Due totheir relatively high stability and compatibility with dry conditions,oligonucleotide polymers such as primers and/or probes that take part inbiological assays are often an ideal choice for predisposal intoindividual process arrays.

One method for predisposal of oligonucleotide components of a biologicalassay into a microfluidic device comprises adding a small volume ofliquid containing oligonucleotide components to each process chamber ina component of a microfluidic device containing the process chambersthat is subsequently assembled into the microfluidic device. To ensureagainst cross contamination of oligonucleotide components betweenprocess chambers, a multichannel liquid handling device may be used suchthat each process chamber corresponds to a separate liquid handling portor tip in the manufacturing system.

As an example, a 384-process chamber sample processing device may haveoligonucleotide components disposed in each process chamber by the useof a 384 channel liquid handling device. Often, the liquids on thesedevices are handled through plastic tips. These tips are typicallymanufactured of hydrophobic plastic polymer materials, such aspolypropylene.

Nucleic acids sometimes demonstrate a propensity to adhere tohydrophobic surfaces of this type. Because of this property, achieving ahigh level of accuracy of delivery of a precise concentration and volumeof an oligonucleotide solution to multiple process chambers in amicrofluidic device, or other device composed of wells, is difficult andcostly. As throughput of manufacturing of these devices with predisposedoligonucleotide components is increased, these effects are magnified tothe point that the oligonucleotide assays to be performed with thedevice are adversely affected. What is needed, then, is a method thatenables large scale production of microfluidic devices, or otherdevices, containing predisposed oligonucleotide components with a highlevel of accuracy and reproducibility. Such methods are provided by thepresent invention.

In certain embodiments, a low percentage of a nonionic detergent isadded to an oligonucleotide spotting solution. A preferred spottingsolution contains the oligonucleotide components of an nucleic aciddetection assay, and optionally a known quantity of a tracer dye tocontrol for manufacturing variability.

In some embodiments, a tracer dye comprises a free label present at aknown concentration. In some embodiments this free label comprises afluorophore. In some other embodiments, the tracer dye comprises a shortoligonucleotide of known sequence that is linked to a label and presentat a known concentration. In some other embodiments, the tracer dyecomprises a mixture of both free label and oligonucleotide-linked label,both of which are present at known concentrations.

The present invention is not limited by the non-ionic detergentemployed. Suitable non-ionic detergents can be found by screeningcandidate non-ionic detergents using the methods described in Example 8by substituting in the candidate non-ionic detergent for those recitedin Example 8. Examples of non-ionic detergents that may be employed,include, but are not limited to: polyoxyethylene surfactants, carboxylicester surfactants, carboxylic amide surfactants, n-dodecanoylsucrose,n-dodecyl-β-D-glucopyranoside, n-octyl-β-D-maltopyranoside,n-octyl-β-D-thioglucopyranise, n-decanoylsucrose,n-decyl-β-D-maltopyranoside, n-decyl-β-D thiomaltoside,n-heptyl-β-D-glucopyranoside, n-heptyl-β-D-thioglucopyranoside,n-hexyl-β-D-glucopyranoside, n-nonyl-β-D-glucopyranoside,n-octanoylsucrose, n-octyl-β-D-glucopyranoside,cyclohexyl-n-hexyl-β-D-maltoside, cyclohexyl-n-methyl-β-D-maltoside,digitonin, and those available under the trade designations PLURONIC,TRITON, TWEEN, as well as numerous others. Without limitation, certainpreferred surfactants of the present invention are the hydroxyethoxyethers, such as TWEEN.

In some embodiments of the invention, the concentration of the nonionicdetergent makes up less than 1% of solution by volume of the nucleicacid mixture. In certain embodiments, the concentration of the nonionicdetergent makes up between 0.001 and 0.1% of the solution by volume ofthe nucleic acid mixture. In other embodiments, the nonionic detergentis present in the nucleic acid mixture spotting solution at aconcentration of 0.005% by volume.

In certain embodiments, the nucleic acid mixture with the non-ionicdetergent is dispensed into the wells of a microfluidic sample device orcomponent thereof. Numerous microfluidic sample processing devices areknown in the art. Examples of such devices, and methods for making andusing such devices, are described in the following patents andapplications: U.S. Pat. No. 6,627,159; U.S. Pat. No. 6,720,187; U.S.Pat. No. 6,734,401; U.S. Pat. No. 6,814,935; U.S. Application2002/0064885; and U.S. Application 2003/0152994; all of which are hereinincorporated by reference for all purposes.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); DS (dextran sulfate); and ° C. (degrees Centigrade).

Example 1 Qualitative Detection of Low Copy Numbers of Viral RNA

This example describes the qualitative detection of low copy number ofviral RNA employing extended rounds of PCR and two INVADER assays thatdetect different parts of the viral RNA, but report to the same FRETcassette. Oligonucleotides were prepared and mixed as follows. Forwardand reverse primers for a first viral target region and a second viraltarget region were generated, as well as forward and reverse primers forthe universal internal control (UIC). All of the reverse primers weredesigned to also serves as INVADER oligonucleotides for their respectivetarget regions. All of these primers were provided at a finalconcentration of 0.5 uM. Primary probes for the first and second viraltarget regions were generated, with each probe containing a 3′ portionspecific for either the first or second viral target region, and 5′flaps that were identical as designed to hybridize to a first FRETcassette (labeled with FAM) provided in the oligonucleotide mixture. Aprimary probe specific for the UIC was also generated that contained a3′ region specific for the UIC control, and a 5′ region that reported asecond FRET cassette (labeled with Yellow dye). All of the primaryprobes were provided at a final concentration of 0.667 uM and both FRETcassettes were provided at a final concentration of 0.25 uM.

The enzyme mixture was prepared as follows: 13.36 ng/μL Cleavase® VIII,0.034 Units/μL native Taq DNA polymerase (Promega), 2 Units/μL MMLV RT(Promega), 0.1 mM DTT. The enzyme mix, target nucleic acid, 20 ng/μLtRNA, and oligonucleotides were mixed in Cleavase® dilution buffer (20mM Tris pH 8.0, 50 mM KCl, 0.5% Tween 20, 0.5% Nonidet P40, 50%glycerol, 0.1 mg/ml BSA). The reactions were performed a buffercontaining 10 mM MOPS, 7.5 mM MgCl2, and 25 μM dNTPs. Universal internalcontrol template (5′-CCCUGCAACGCGAGUGCUGAGGCUGGUGUACGACCCAUCGCUCGCCCGCUACCGCGACGUCCUGCCGCACUCUAGGUACGUGGUCCAC-3′, SEQ ID NO:5) waspresent at 150 copies per reaction, or omitted, as shown in the results.Viral template RNA was prepared by extraction of virion (Acrometrix) RNAwith the high pure RNA extraction kit (Roche) according to themanufacturer's instructions, and added to the reaction mixture atconcentrations ranging from 2.06 to 500 copies per reaction, as shown inthe results. All the aforementioned reagents were mixed into 25 μLreactions, and subjected to temperature incubations as follows: 42° C.for 30 min, 95° C. for 2.5 min, 35 cycles at (95° C. for 20 sec and 72°C. for 1 min), 99° C. for 10 min, and 58° C. for 30 min. The fluorescentsignal produced in each reaction vessel was quantitated on a TecanGenios FL fluorescence plate reader.

As shown in FIG. 1, the above assay was able to detect viral RNAtemplate present in the reaction vessel when present at approximately 2copies per reaction. The UIC was detected at approximately equivalentlevels in each reaction, with some minor fluorescent signal cross-talkapparent in the reactions containing the highest levels of targettemplate. This Example demonstrates how hard to detect viral RNAsequence, present at very low levels, can be detected by amplifying tworegions of the viral RNA with extended rounds of PCR and detecting eachtarget region with an invasive cleavage assay that reports to the samereadout channel (e.g. same FRET cassette).

Example 2 Quantitative Detection of a Broad Range of Copy Numbers ofViral RNA

The following example describes the use of two probes at differentconcentrations that each contributes to extend the dynamic range ofdetection of a viral RNA target sequence using a single dye fordetection. In this example, a different FRET cassette was provided toaccumulate signal from each probe, but the FRET cassettes reported usingthe same dye.

Oligonucleotides were prepared and mixed in three different pools: “1×”,“0.01×”, and “1×+0.01×”. The following oligonucleotides were used.Forward and reverse primers for a particular viral target region weregenerated, as well as forward and reverse primers for the universalinternal control (UIC). All of the reverse primers were designed to alsoserves as INVADER oligonucleotides for their respective target regions.All of these primers were provided at a final concentration of 0.5 uM.First and second primary probes were generated having identical 3′regions specific for the target viral RNA and 5′ flaps that weredifferent. The flaps were designed to hybridize to different FRETs(first and second FRETs), but the FRETs were designed with the same dye(red dye). A third primary probe specific for the UIC was also generatedthat contained a 3′ region specific for the UIC control, and a 5′ regionthat reported a third FRET cassette (labeled with Yellow dye). The firstand second primary probes were provided at different concentrations. Thefirst primary probe was provided at a final concentration of 0.667 uMand the second primary probe was provided at a final concentration thatwas 100-fold lower (i.e. 0.0067 uM). The third primary probe wasprovided at the standard concentration of 0.667 uM. All of the FRETcassettes were provided at a final concentration of 0.25 uM.

The enzyme mixture for this example was prepared as follows: 13.36 ng/μLCleavase® VIII, 0.034 Units/μL native Taq DNA polymerase (Promega), 2Units/μL MMLV RT (Promega), 0.1 mM DTT. The enzyme mix, target nucleicacid, 20 ng/μL tRNA, and oligonucleotides were mixed in Cleavase®dilution buffer (20 mM Tris pH 8.0, 50 mM KCl, 0.5% Tween 20, 0.5%Nonidet P40, 50% glycerol, 0.1 mg/ml BSA). The reactions were performedin a buffer containing 10 mM MOPS, 7.5 mM MgCl2, and 25 μM dNTPs.Universal internal control template (SEQ ID NO:5) was present at 66.7copies per reaction, or omitted, as shown in the results. Viral templateRNA was prepared by extraction of virion (Acrometrix) RNA with the highpure RNA extraction kit (Roche) according to the manufacturer'sinstructions and added to the reaction mixture at concentrations rangingfrom 50 to 160,000,000 copies per reaction, as shown in the results. Allthe aforementioned reagents were mixed into 50 μL reactions, andsubjected to temperature incubations as follows: 42° C. for 30 min, 95°C. for 2.5 min, 25 cycles at (95° C. for 20 sec and 72° C. for 1 min),99° C. for 10 min, and 58° C. for 30 min. The fluorescent signalproduced in each reaction vessel was quantitated on a Tecan Genios FLfluorescence plate reader.

As shown in FIG. 2, the assay was able to detect viral RNA in a lineardynamic range from 50 to 8,000,000 copies, when the “1×+0.01×”oligonucleotide mixture was used. The “1×” and “0.01×” lines depict thesignal generated when the “1×” or “0.01×” oligonucleotide mixtures wereused independently. This experiment demonstrates using multiple probesat different concentrations is one way to increase dynamic range whendetecting viral RNA.

Example 3 Detecting Two Different Types of Viral Nucleic Acid in SingleReaction Vessel Using Invasive Cleavage Assays that Report to DifferentDyes

This Example describes detecting two different types of viral nucleicacid in a single reaction vessel using invasive cleavage assays thatreport different colors.

Oligonucleotides were prepared and mixed as follows. Forward and reverseprimers for a first viral target RNA sequence and a second viral targetDNA sequence, with the second viral target being a viral nucleic acidsequence from a different type of virus than the first viral targetsequence, were generated. Both of the reverse primers were designed toalso serves as INVADER oligonucleotides for their respective targets.All of these primers were provided at a final concentration of 1.0 uM.Primary probes for the first and second viral target sequences weregenerated, with each probe containing a 3′ portion specific for eitherthe first or second viral target sequence, and 5′ flaps that weredifferent from each other and designed to different FRET cassettes. Bothprimary probes were provided at a final concentration of 0.667 uM. Thetwo FRET cassettes were also provided in the oligonucleotide mixture andwere labeled differently, with the first FRET cassette having a FAM dyeand the second FRET cassette having a Red dye. Both FRET cassettes wereprovided at a final concentration of 0.334 uM.

The enzyme mixture was prepared as follows: 6.7 ng/μL Cleavase® VIII,0.02 Units/μL native Taq DNA polymerase (Promega), 2 Units/μL MMLV RT(Promega). The enzyme mix, target nucleic acid, 20 ng/μL tRNA, andoligonucleotides were mixed in reactions performed in a buffercontaining 10 mM MOPS, 7.5 mM MgCl₂, and 25 μM dNTPs. The first viraltarget sequence template RNA was prepared by extraction of virion(Acrometrix) RNA with the high pure RNA extraction kit (Roche) accordingto the manufacturer's instructions and added to the reaction mixture atconcentrations ranging from approximately 2 to 62 copies per reaction,as shown in the results. The second viral target sequence template DNAextracted from virion (Advanced Biotechnologies) was added to thereaction mixture at concentrations ranging from approximately 35 to 1116copies per reaction, as shown in the results. All the aforementionedreagents were mixed into 100 μL reactions, and subjected to temperatureincubations as follows: 42° C. for 30 min, 95° C. for 5 min, 28 cyclesat (95° C. for 45 sec, 69.5° C. for 45 sec and 72° C. for 90 sec), 99°C. for 10 min, and 58° C. for 15 min. The fluorescent signal produced ineach reaction vessel was quantitated on a Tecan Genios FL fluorescenceplate reader.

As shown in FIG. 3, first virus-derived target RNA and secondvirus-derived target DNA were simultaneously detected in the samereaction vessel using the methods described in this Example, withdistinguishing fluorescent signal directed to the FAM and Redfluorescent channels, respectively.

Example 4 Detecting Two Different Types of Viral Nucleic Acid in SingleReaction Vessel Using Invasive Cleavage Assays that Report to the SameDye

This Example describes detecting two different types of viral nucleicacid in a single reaction vessel using invasive cleavage assays thatreport to the same dye. Oligonucleotides were prepared and mixed asfollows. Forward and reverse primers for a first viral target RNAsequence and a second viral target DNA sequence, with the second viraltarget being a viral nucleic acid sequence from a different type ofvirus than the first viral target sequence, were generated. Both of thereverse primers were designed to also serves as INVADER oligonucleotidesfor their respective targets. All of these primers were provided at afinal concentration of 1.0 uM. Primary probes for the first and secondviral target sequences were generated, with each probe containing a 3′portion specific for either the first or second viral target sequence,and 5′ flaps that were different from each other and designed todifferent FRET cassettes. Both primary probes were provided at a finalconcentration of 0.667 uM. The two FRET cassettes were also provided inthe oligonucleotide mixture and were labeled with the same dye (bothwith FAM dye). Both FRET cassettes were provided at a finalconcentration of 0.334 uM.

The enzyme mixture was prepared as follows: 6.7 ng/μL Cleavase® VIII,0.02 Units/μL native Taq DNA polymerase (Promega), 2 Units/μL MMLV RT(Promega). The enzyme mix, target nucleic acid, 20 ng/μL tRNA, andoligonucleotides were mixed in reactions performed in a buffercontaining 10 mM MOPS, 7.5 mM MgCl2, and 25 μM dNTPs. First viraltemplate RNA was prepared by extraction of virion (Acrometrix) RNA withthe high pure RNA extraction kit (Roche) according to the manufacturer'sinstructions and added to the reaction mixture at concentrations rangingfrom approximately 2 to 62 copies per reaction, as shown in the results.Second viral template DNA was extracted from virion (AdvancedBiotechnologies) and was added to the reaction mixture at concentrationsranging from approximately 35 to 1116 copies per reaction, as shown inthe results. All the aforementioned reagents were mixed into 100 μLreactions, and subjected to temperature incubations as follows: 42° C.for 30 min, 95° C. for 5 min, 28 cycles at (95° C. for 45 sec, 69.5° C.for 45 sec and 72° C. for 90 sec), 99° C. for 10 min, and 58° C. for 15min. The fluorescent signal produced in each reaction vessel wasquantitated on a Tecan Genios FL fluorescence plate reader.

As shown in FIG. 4, first virus-derived target RNA and secondvirus-derived target DNA were simultaneously detected in the samereaction vessel using the methods described in this Example, withdetection signal reported to the same fluorescent channel.

Example 5 Detection of Viral RNA Using Multiple Stem Oligonucleotides

This example describes the use of stem oligonucleotides to detect viralRNA directly using the INVADER assay. Combining the stemoligonucleotides with the downstream probe and upstream oligonucleotideallows a T-structure to form as shown with the hypothetical sequences inFIG. 6. This allows a “T-structure INVADER assay” to be performed.

In this example, three sets of oligonucleotides were designed that eachanneal to specific regions of the same viral RNA genome. The threeregions of the target viral RNA were designated Region 1, Region 5, andRegion 8. Each stem oligonucleotide contained the same probe annealingregion so that each INVADER cleavage structure would release the sameprobe flap upon cleavage by CLEAVASE and could be detected by a singlesecondary FRET cassette containing oligonucleotide. In this example,each of the sets was used separately, and two of the stemoligonucleotide sets were used simultaneously.

The T-structure INVADER assay was used to detect both purified viral RNAand viral RNA from an viral RNA-positive serum sample. The T-structureINVADER assay reactions were set up as follows: In a 96-well plate, to a10 uL sample was added 5 uL of 4× CLEAVASE buffer (containing 40 mM MOPSBuffer and 56 mM MgCl₂), 4 uL of 5× oligonucleotide mixture (containing2.5 uM downstream probe oligonucleotide, 1 nM upstream (INVADER)oligonucleotide, 1 nM Stem oligonucleotide, and 1.25 uM FRET cassetteoligonucleotide), and 1 uL of Cleavase X (at 40 ng/uL). This mixture wasincubated at 75° C. for 5 minutes, then at 63° C. for 4 hours. Followingthis incubation, fluorescent signal from the reaction was detected in amicroplate fluorimeter.

The upstream oligonucleotides were blocked on their 5′ ends with2′-O-methylated bases, while the stem oligonucleotides were blocked onboth their 5′ and 3′ ends with 2′-O-methylated bases. Three differentregions on the same viral RNA target sequence were detected. For each ofthe three targets, a particular stem oligonucleotide and particularupstream oligonucleotide were employed, while all three targets employedthe same downstream probe. In regard to the three stem oligonucleotides,each of these oligonucleotides had a particular 3′ target specificregion, and a particular stem region, where the stem region wasconfigured to hybridize to the hybridize to a portion of both theupstream oligonucleotide and the downstream probe. In regard to thethree upstream oligonucleotides, each of these had a 5′ target specificregion, a stem specific region configured to hybridize to a portion ofthe stem region of the stem oligonucleotide, and a 3′ region (one basein this example) configured to not hybridize to the stemoligonucleotide. The general T-structure configuration formed by thecombination of these sequences at each target site is shownschematically in FIG. 6 (which shows a hypothetical target sequence).

In this Example, Invader/Stem/Probe assays for regions 1 and 8 weretested separately and together to detect purified viral RNA and viralRNA from viral RNA-positive plasma. The results are shown below inTables 1 and 2.

TABLE 1 Viral RNA Transcripts 4 hr incubation: Copy number in assay Site1 Site 8 Site 1 + Site 8 Raw No Target Control 354 350 363 Signal 500004341 4496 4405 Count 18000 1058 1439 1889 6000 630 753 968 2000 449 502574 1000 399 458 492 500 392 434 445 Signal 50000 12.26 12.85 12.13 Fold18000 2.99 4.11 5.20 Over 6000 1.78 2.15 2.67 Control 2000 1.27 1.431.58 1000 1.13 1.31 1.36 500 1.11 1.24 1.23

TABLE 2 Viral RNA extracted from viral-positive plasma 4 hr incubation:Copy number in assay Site 1 Site 8 Site 1 + Site 8 Raw Negative Plasma357 369 366 Signal 140000 2643 3007 3687 Count 42000 1466 1492 223514000 719 747 1092 4200 482 509 629 1400 414 433 468 Signal 140000 7.408.15 10.07 Fold 42000 4.11 4.04 6.11 Over 14000 2.01 2.02 2.98 Control4200 1.35 1.38 1.72 1400 1.16 1.17 1.28Results for the T-structure INVADER assay for region 5 are shown belowin Table 3.

TABLE 3 Copy number in assay 4 hr incubation Raw No Target Control 310Signal 18000 1006 Count 6000 522 2000 571 Signal 18000 3.25 Fold Over6000 1.68 Control 2000 1.84

Example 6 Optimization of Oligonucleotide Concentration

In this Example, oligonucleotide concentrations were optimized for usein the T-structure INVADER assay. Reactions were set up as in Example 5above, but Region 1 upstream (INVADER) and Stem oligonucleotideconcentrations were varied from 50 nM to 1 pM. 50,000 copies of viralRNA transcripts were used as the template. Results are shown below inTable 4.

TABLE 4 50 nM 5 nM 0.1 nM 1 pM Raw Signal Count No Target 4492 1554 310296 Control target 4872 2692 471 300 transcript Signal Fold target 1.081.73 1.52 1.01 Over Control transcript

Example 7 Optimization of Length of Stem Specific Region of UpstreamOligonucleotide

In this Example, the length of the upstream (INVADER) oligonucleotide,and particularly the stem specific region of the upstreamoligonucleotide, was optimized for use in the T-structure INVADER assay.The experiments were performed as above, using viral RNA transcripts asthe template, with the exception that the INVADER reaction was incubatedfor 6 hours. Upstream oligonucleotides with 5 base, 9 base, and 12 basestem specific regions were tested. As shown in Table 5 below, upstreamoligonucleotides with t9 base stem specific regions performed betterthan the 5 base and 12 base stem specific regions.

TABLE 5 Copy number in assay 5 bases 9 bases 12 bases Raw Signal NoTarget Control 297 320 380 Count 24,000 347 518 486 8,000 302 441 470Signal Fold 24,000 1.17 1.62 1.28 Over Control 8,000 1.02 1.38 1.24

Example 8 Use of Nonionic Detergent to Increase Accuracy ofOligonucleotide Spotting

The following example describes the use of nonionic detergent toincrease the reproducibility and accuracy of spotting of oligonucleotideassay components on a microfluidic device component.

In this example, oligonucleotide components of an oligonucleotidedetection assay are added to a sheet of molded plastic that contains thedevice process chambers which will later be manufactured into a 384-wellmicrofluidic device after adhesion to a metal foil backing. In order tocontrol for manufacturing variability, certain spots in each deviceinclude a synthetic “tracer dye” which is composed of red fluorophorecovalently linked to an oligonucleotide dT10 molecule. Certain spots ineach device also contain a free green fluorophore as an alternativemeans of manufacturing control. The fluorescence of the red tracer dye,as an indicator of the quantity of tracer added to each device,correlates closely with the performance of the oligonucleotide detectionassay in downstream applications.

The final concentration of the components of the oligonucleotidespotting solutions in this Example were as follows: 17 mM MOPS(3-(N-morpholino) propanesulfonic acid), 240 nM of oligonucleotidetracer dye, and 10 ng/mL free fluorophore.

In the context of high-throughput manufacturing, an unacceptable levelof device-to-device variability resulted (FIG. 8A). In thismanufacturing protocol, a 384-tip liquid handling robot, usingNanoScreen Tips, aspirates sufficient volume from a master plate to add1 uL per spot to each of 24 microfluidic devices in series. This cycleof 24 devices is repeated 5 times, with 5 aspiration steps from the samemaster plate, for spotting of a total of 120 devices in eachmanufacturing run. As shown in FIG. 8A, the fluorescent signal detectedfrom the Red tracer dye increases significantly towards the first andlast devices of each cycle of 24, with the magnitude of this cyclicaltrend itself increasing with each subsequent round of 24 devices up to120. It is also noteworthy that the signal from the green freefluorophore, which serves as an indicator of the total volume added toeach spot, was more or less consistent throughout the application, whilethe signal from the red oligonucleotide tracer dye, which serves as anindicator of the actual concentration of dye added to each spot, showeda high degree of variability (FIG. 8A). This suggested that the sourceof the increasing variability was associated with a change in the actualconcentration of the oligonucleotide components being added to each spotin series. Although the reason for the change in concentration over timeis not known, one possibility is that the oligonucleotide componentswere adhering to the polypropylene materials used in the tips of theliquid handling device, which would in turn affect the actualconcentration of oligonucleotide tracer being added to each spot duringmanufacturing.

First, two varieties of liquid handling tips, NanoScreen tips andBeckman tips, both made from similar polymer materials, were tested. Theresults of this experiment demonstrated that the problem of spottingvariability were not associated with the peculiarities of any singletype of liquid handling type, but rather more generally to theproperties of the materials the liquid handling tips are produced from.FIG. 8A shows the results using NanoScreen tips and FIG. 8B shows theresults using Beckman tips.

Two hypothesis were tested to determine whether additional materialsadded to the spotting solution could solve the problems of spottingvariability. In the first, transfer RNA (tRNA) was used. It is wellknown in the art of oligonucleotide properties that mixtures ofadditional nucleic acids can often serve to “block” the nonspecificbinding of the desired oligonucleotides to a variety of surfaces. Onecommon type of blocking nucleic acid used is a preparation of tRNAmolecules commonly available for this purpose. A solution of Brewer'sYeast tRNA at a final concentration of 94 μg/mL was added to theoligonucleotide spotting mixture, and the same type of 120 plate cycle(5 runs with 24 plates spotted in each run) was run as described abovewith free fluor and red tracer. The results are shown in FIGS. 9B (withthe added tRNA) and 9A (control containing a water diluent instead oftRNa). The results of this experiment indicated that the use of blockingnucleic acids would not function to prevent spotting variability ingenerating pre-loaded microfluidic devices.

A second set of assays were conducted to test the effect of nonionicdetergents on spotting variability. In the first experiments, twononionic detergents (Tween-20 and Nonidet P40, a.k.a. NP40) were testedin the context of the oligonucleotide spotting solution. As shown inFIG. 10B, the incorporation of the nonionic detergents led to thecomplete diminution of the problem of spotting variability inmanufacturing of pre-loaded microfluidic devices. FIG. 10A shows acontrol that used a water diluent and no non-ionic detergents.

The contribution and minimum concentration of each nonionic detergentwas subsequently tested. Both Tween-20 and NP40 worked equally well inthis system. FIG. 11A shows a control using water; FIG. 11B shows theresults using 0.25% TWEEN; FIG. 11C shows the results using 0.005%TWEEN; and FIG. 11D shows the results using 0.0025% TWEEN. As shown inthese figures, the minimum concentration of nonionic detergent requiredto provide consistent spotting performance was around 0.005%. Theseresults demonstrate that the use of nonionic detergent in theoligonucleotide spotting mixture helps eliminate spotting variabilityproblems associated with high throughput microfluidic devicemanufacturing.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described compositions and methods of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin the relevant arts are intended to be within the scope of thefollowing claims.

1. A method of target nucleic acid dependent amplification of anon-target sequence in a sample comprising; a) incubating a sample withstem oligonucleotides, upstream oligonucleotides, primers, dNTPs, and apolymerase under conditions such that, if the target nucleic acid ispresent: i) a 3′ target specific region of said stem oligonucleotideshybridizes to said target nucleic acid, and a stem region of said stemoligonucleotides remains available for hybridization to said upstreamoligonucleotides, ii) a 5′ target specific region of said upstreamoligonucleotides hybridizes to said target nucleic acid, and a stemspecific region of said upstream oligonucleotides hybridizes to aportion of said stem region of said stem oligonucleotides, and iii) saidpolymerase extends the 3′ end of said upstream oligonucleotides usingsaid stem region of said stem oligonucleotides as a template to generateextended upstream oligonucleotides that comprise an upstreamoligonucleotide extended region; b) heating said sample in order toseparate said extended upstream oligonucleotides from said stemoligonucleotides and said target nucleic acid; c) cooling said sampleunder conditions such that said primers hybridize to at least a portionof said upstream oligonucleotide extended region of said extendedupstream oligonucleotides; and d) incubating said sample underconditions such that said primers are extended by said polymerase usingsaid extended upstream oligonucleotide as a template such that stemamplicon sequences are generated.
 2. The method of claim 1, furthercomprising the step of detecting the presence or absence of said targetnucleic acid in said sample by determining if stem amplicon sequencesare generated.
 3. The method of claim 1, further comprising the step ofperforming one or more rounds of PCR using said stem amplicon sequencesand said extended upstream oligonucleotides as templates, wherein saidupstream oligonucleotides prime polymerization from said stem ampliconsequences, and wherein said primers prime polymerization from saidextended upstream oligonucleotides.
 4. The method of claim 3, furthercomprising a step of detecting the presence or absence of said targetnucleic acid in sample sample by detecting any accumulated PCR products.5. The method of claim 1, further comprising incubating said sample withdownstream probes and upstream oligonucleotides such that invasivecleavage structure are formed with said stem amplicon sequences, thedownstream probes, and the upstream oligonucleotides.
 6. A methodcomprising: incubating a sample with stem oligonucleotides, upstreamoligonucleotides, dNTPs, and a polymerase under conditions such that, ifthe target nucleic acid is present: A) a 3′ target specific region ofsaid stem oligonucleotides hybridizes to said target nucleic acid, and astem region of said stem oligonucleotides remains available forhybridization to said upstream oligonucleotides, B) a 5′ target specificregion of said upstream oligonucleotides hybridizes to said targetnucleic acid, and a stem specific region of said upstreamoligonucleotides hybridizes to a portion of said stem region of saidstem oligonucleotides, and C) said polymerase extends the 3′ end of saidupstream oligonucleotides using said stem region of said stemoligonucleotides as a template to generate extended upstreamoligonucleotides that comprise an upstream oligonucleotide extendedregion.
 7. The method of claim 6, further comprising the step of heatingsaid sample in order to separate said extended upstream oligonucleotidesfrom said stem oligonucleotides and said target nucleic acid.
 8. Themethod of claim 7, further comprising the step of cooling said sampleunder conditions such that said primers hybridize to at least a portionof said upstream oligonucleotide extended region of said extendedupstream oligonucleotides.
 9. The method of claim 8, further comprisingthe step of incubating said sample under conditions such that saidprimers are extended by said polymerase using said extended upstreamoligonucleotide as a template such that stem amplicon sequences aregenerated.
 10. The method of claim 9, further comprising the step ofdetecting the presence or absence of said target nucleic acid in saidsample by determining if stem amplicon sequences are generated.
 11. Themethod of claim 9, further comprising the step of performing one or morerounds of PCR using said stem amplicon sequences and said extendedupstream oligonucleotides as templates, wherein said upstreamoligonucleotides prime polymerization from said stem amplicon sequences,and wherein said primers prime polymerization from said extendedupstream oligonucleotides.
 12. The method of claim 11, furthercomprising a step of detecting the presence or absence of said targetnucleic acid in said sample by detecting any accumulated PCR products.13. The method of claim 9, further comprising incubating said samplewith downstream probes and upstream oligonucleotides such that invasivecleavage structure are formed with said stem amplicon sequences, thedownstream probes, and the upstream oligonucleotides.